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BIOSENSORS: Unit 1: Biosensors Functioning. What is a biosensor? ―A biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi quantitative analytical information using a biological recognition element (bioc...

BIOSENSORS: Unit 1: Biosensors Functioning. What is a biosensor? ―A biosensor is a self-contained integrated device which is capable of providing specific quantitative or semi quantitative analytical information using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transduction element‖. Key Words:  self-contained integrated device.  specific quantitative or semi-quantitative analytical information.  biological recognition element.  transduction element.  direct spatial contact – Surface Functionalization. First Approximation: Definition: A biosensor is a device used for the detection of biological elements. The detection is based on biomolecule specific recognition events. Parts of a biosensor:  Biological element for specific detection (Bioreceptor).  Transductor.  Electronic device to get and analyze the obtained signal. A little bit of history: 1) Ancient Egypt: Papiro de Ebers (1553 a.C): Animal behaviours and indicators of anomalies in the urine composition – Today diabetes. 2) The primitive “CANARIOS” used in “MINAS” to detect the presence of lethal gases can be considered a primitive example of biosensor. 3) Chemistry and Biochemistry Development: Classical analytic methods (not biosensors): Need a big sample; Slow; Qualified personal; Time of analysis. 1  Leland C. Clark (Father of the biosensors): Voltamperommetry: Reference electrode to calibrate the main one; Covered in plastic and glass – reduced oxygen consumption. Clark type electrode for measuring oxygen: A) Platinum electrode; B) Silver electrode that acts as a reference; C) Potassium chloride, electrolyte; D) Teflon membrane that permeably isolates the electrodes so as not to consume much oxygen; E) Rubber ring; F) Battery; G) Galvanometer to measure potential variations dependent on oxygen concentration. In this electrode the O2 is reduced in the Pt cathode, and the Ag is oxidated freeing electrons that generate electric current that is measurable and proportional to oxygen. ½ O2 + H2O + 2e-  2OH Ag + Cl-  AgCl + e- Clark and Lyons indicated the possibility to improve the electrochemical usual sensors using enzymes trapped in a semipermeable membrane. This concept was illustrated with an experiment in which the enzyme glucose oxidase was confined near the electrode by a dialysis membrane, observing that its response was proportional to the glucose concentration. In this publication, Clark and Lyons coined the term "enzymatic electrode" to describe this type of biosensor, which was later implemented by Updike and Hicks. In 1975, it was successfully commercialized by the company Yellow Springs Instrument (Ohio), as a glucose analyser based on the amperometric detection of hydrogen peroxide. One of the most famous: Glucose Amperometric Biosensor: Uses an enzyme that processes the glucose molecules, liberating an electron for each processed molecule. This electron is captured by an electrode and the electron flux is used as a measure of the glucose concentration. 2 Difference between biosensors, chemical sensors and sensors for biological signals: A chemical sensor is a device that transforms chemical information, ranging from the concentration of a specific sample component to total composition analysis, into an analytically useful signal. A wide range of materials has been developed in order to simulate the biological functions. A great advantage of the chemical sensors is that their production is more affordable than biosensor‘s and that they can be applied in environments where the bio components lose their activity, such as high pH or temperatures changes. However, and as we saw, they need specially trained personal, time, big sample, etc. Chemical (or others) sensors differ from biosensors by the fact that they don’t use a biological component in the signal transduction. A biosensor is an analytical device that measures biological or chemical reactions, containing a biological receptor, warranting specificity and producing a signal translated by a chemical or physical component. Elements of a Biosensor: “Analyte”: A substance of interest that needs detection (the target). It is going to bind to the bioreceptor. Examples of analytes: Glucose, pH, Lactate, Alcohol, Urea, Cholesterol, DNA, Proteins, Antibody/antigen, Neurotransmitters, Toxins, Antibiotics, Vitamins, Environmental Pollutants, Ions/Cations, Gases, Hormones. Bioreceptor: A molecule that specifically recognizes the analyte is known as a bioreceptor. The process of signal generation (in the form of light, heat, pH, charge or mass change, etc.) upon interaction of the bioreceptor with the analyte is termed bio-recognition. It has to be immobilized in the surface of the electrode. Function  Detection of specific molecules or biologic systems (target). Examples: Antibody/Antigen, Proteins, Nucleotide chains – DNA, Ionophore, Microorganisms: viruses, bacteria, Enzymes, Cell Organelle, Whole Cell, Tissue, Biomimetic systems, Part of an organ, Biomembranes.  Affinity biosensors: when the bioreceptor just binds to the analyte. Their functioning is based in the interaction of the analyte with the recognizing element (there is not a catalytic transformation). They have a noncatalytic bond. (No reaction is produced. Ex: Antibodies, nucleic acids including DNA, proteins, molecularly imprinted polymers MIPs). 3   Catalytic biosensors: Affinity + Catalysis: The biological element combines with the analyte. The bioreceptors are catalyzers which force a chemical reaction. Even with the behavior of products and reactives, or by enzymatic mechanisms (in which the enzymes are not consumed). Exa.: Enzymes, cells, organelles and tissue (this three can be used also in affinity biosensors). Caution! The type of analyte-bioreceptor bonding is different from the bioreceptor-biosensors surface bonding. Transducer: The transducer is an element that converts one form of energy into another. In a biosensor the role of the transducer is to convert the bio-recognition event into a measurable signal. This process of energy conversion is known as signalisation. Most transducers produce either optical or electrical signals that are usually proportional to the amount of analyte–bioreceptor interactions. Function  Transform the specific (biological) reaction between the probe and the analyte or target into some type of signal that can be read, analyzed and quantified (physical). Requirements: Work in adequate physical-chemical conditions Types  Optics: reflectance, flurescence Mechanical Electrical: piezoresistive, piezoelectric, electrochemical Thermometric. Electronics: The part of a biosensor that processes the transduced signal and prepares it for display. It consists of complex electronic circuitry that performs signal conditioning such as amplification and conversion of signals from analogue into the digital form. The processed signals are then quantified by the display unit of the biosensor. Display: The display consists of a user interpretation system such as the liquid crystal display of a computer or a direct printer that generates numbers or curves understandable by the user. This part often consists of a combination of hardware and software that generates results of the biosensor in a user- friendly manner. The output signal on the display can be numeric, graphic, tabular or an image, depending on the requirements of the end user. Typically composed by set of cards , computer programs and PCs. Types of biosensors: A) Electrochemical Biosensors: The first developed biosensors. They measure electrochemical changes produced due to the interaction between the analyte and the bioreceptor. 1. Variation in the analyte/product/subproduct concentration. 2. Changes in the surface of the electrode. They are usually used with biocatalytic bioreceptors, because they cause or consume electroactive species, producing changes in the pH, the potential, electrical resistance, etc. Exa.: Amperometry; Potentiometry; Conductimetry (and impedimetric); Field-effect transistors (FETs). 4  B) Optic Biosensors: They measure the variations in the properties of an electromagnetic radiation as consequence of the interaction between the analyte and the bioreceptor. The changes in the optical properties induced by the molecular recognition reaction when the electromagnetic radiation hit the surface where the bioreceptor is immobilized. Exa.: Optic fiber sensors (Optrodes); Plasmon Surface Resonance (SPR); Mirror resonance; Interferometers; Total internal reflection fluorescence. C) Mechanical Biosensors: They are based in mobile or flexible structures that suffer a deflexion in one of its edges due to the incorporation of the analyte. Exa.: Static mode: changes in the position; Dynamic mode: changes in movement. D) Acoustic or mass Biosensors: They are based in piezoelectric crystals that are subjected to an external electrical signal that makes them vibrate at their resonance frequency. They are based in the piezoelectric effect by which certain materials generate an electric current when mechanically deformed. Therefore, they measure mass changes in the surface of the biosensor. Exa.: SAW (Surface Acoustic Wave) type; BAW (Bulk Acoustic Wave) type. E) Thermometric Biosensors: They measure temperature changes due to the exothermic biochemical reactions. Being the most common those based in enzymatic catalysis. F) Magnetic Biosensors: They use magnetic nanoparticles to which the bioreceptors are attached and little changes in magnetic fields are measured. Exa.: Magnetometers; SQUIDS. Biosensors Classification: 5 Characteristics of a Biosensor: There are certain static and dynamic attributes that every biosensor possesses. The optimization of these properties is reflected on the performance of the biosensor.  Specificity/Selectivity: Specificity is the ability to assess an exact analyte in a mixture, whereas selectivity is the ability to differentiate analytes in a mixture from each other.  Stability: Degree of susceptibility to ambient disturbances in and around the biosensing system. It directly affects the precision and accuracy of the biosensor.  Measurement Range: range from the minimum and maximum limit of detection that the sensor is expected to measure accurately.  Accuracy: The difference between the true value and the actual value measured by the biosensor.  Precision: The random spread of measured values around the average measured values.  Linearity: Accuracy of the measured response to a straight line. Sensitivity  It refers to the change in output per unit of input variation. It is an essential factor in determining the minimum detectable pressure change. Linear range  Range of analyte concentrations for which the biosensor response changes linearly with the concentration.  Reproducibility: Fast Response, Low cost, Industrially scalable, Reusable, Easy handing, Miniaturization, Small Samples, Easy integration, Multianalysis… 6 While sensitivity refers to the sensor‘s ability to detect small analyte changes, resolution specifically relates to the smallest analyte difference that can be measured and displayed by the sensor. Example: a pressure sensor with a 10 mV/kPa sensitivity and a resolution of 0.1 kPa. This means that the sensor can detect pressure changes as small as 0.1 kPa (resolution), and for each kilopascal increase in pressure, the output voltage will change by 10 millivolts (sensitivity).  Limit of Detection (LoD): The Limit of Detection is defined as when the signal (S) is three times greater than the noise > S/N.  Limit of Quantification (LoQ): The Limit of Quantification is defined as when the signal (S) is ten times greater than the noise > S/N.  Hysteresis: The input-output characteristic follow a different nonlinear trend, depending on whether the input increases or decreases. 7  They have repeated the experiment because of the error lines. The enzyme sticks with agarose. You have to look above all at the slope, so that it does not give you zero, because otherwise the treatment or the biosensor does not respond and the conditions do not matter.  Application of Biosensors: Environmental monitoring, soil quality monitoring, prosthetic devices, food quality monitoring, pathogen discovery, drug discovery, toxin detection, water monitoring, disease detection. Unit 2: Biosensors Surface. Part I. A) Bioreceptors – Biocatalytic Sensors: They use biological catalyzers that drive a chemical reaction in which participate one or several substrates to generate one or several products. When the process is finished, the bio-catalyzer is regenerated and can be reutilized (quantifying the analyte, it is not measured directly from the analyte, so there are two possibilities: we see the disappearance of some substrate, for example oxygen, or you measure one of the products, for example oxygen peroxide). Substrates  Products. * Enzymes can be used over and over again because they regenerate. They can be employed to detect the presence of one of the substrates participating in the reaction in two ways: - Quantifying the disappearance of one co-substrate or known cofactor, different from the one that we want to detect. - Generation of one known product that can be quantified. They can also be used indirectly to detect compounds that interfere with the reaction in a specific way, inhibiting the capacities of the biocatalyzer. Examples - Amperometric Glucose Biosensor: The reaction consumes oxygen. And applying a controlled voltage, the hydrogen peroxide is oxidized at the platinum electrode. The number of electron transfers, at the electrode surface, is directly proportional to the number of glucose molecules present in the blood. (Usually if we have a biocatalytic receptor, the transductors are electrochemical due to the electrons realised in the reaction). Two strategies used for measuring: Measuring the oxygen consumption (Clark) or measuring the amount of hydrogen peroxide produced by the enzyme in the reaction. 8 Examples - Optical Algal Biosensor: The biosensor is constructed to detect heavy metals from inhibition of the enzyme alkaline phosphatase present on the external membrane of Chlorella vulgaris microalgae. The microalgal cells are immobilized on removable membranes placed in front of the tip of an optical fiber. MUP: Methylumbelliferoyl phosphate. MUF: Methylumbelliferone. The reaction product methylumbelliferone (MUF) is fluorescent!! A.1) Enzymes: They catalyze a chemical reaction by the joining of a specific substrate in a specific region on the enzyme. This region is known as the active site. The active site is generally found on the surface of the enzyme. The active site is a three-dimensional cleft, and the substrate are bound to enzymes by multiple weak attractions (electrostatic, hydrogen bond, Van Der Waals forces, Non covalent bond). The enzymatic activity is usually controlled by cofactor presence together with pH and ionic force of the medium where the reaction is going on. An several methodologies are used to increase the stability of the enzymes, as chemical stabilization and/or immobilization. Commercially speaking, the enzymes more used are from the family of oxidoreductases (together with polyphenol oxidases, peroxidases, and aminooxidases). They are stable and do catalyze red-ox reactions being able to be used in different type of transductors: electrochemical, optical, thermometric and piezoelectric ones. They are very selective, fast and the configuration in the prototypes is relatively simple. They present auto- regenerative properties. They are commercially available (very). A.2) Whole cells: Cells precedent from microorganisms or superior organisms: Bacteria, yeast, fungi, algae, and other higher eukaryotes including fish, rat and human cells. According to their cellular activity they can be employed for the analysis of different compounds: - The ones related to cellular growth (vitamins, sugars, organic acids…). - Those that influence in the cell breathing or metabolism in the presence of a toxic or contaminating element. 9 Living cells express a wide variety of molecules (receptors) in different proportions  They can help in quantitatively analyzing more than one analyte while requiring less effort and expenses than other methods  The enzymes and other molecules required for biosensing are present in their native environment and hence display optimal activity and specificity against the target analyte  Thus, cells offer ease in designing the functional strategy of biosensors It is possible use cell-based biosensors for in situ (ex vivo in some cases) monitoring with living cells in their native environment. Living cells in a biosensor are coupled to external transducers (immobilization). Electrochemical and optical are the transductors most commonly used. Cell-based biosensors have evolved into powerful bioelectronic systems that can not only detect specific analytes but can also provide physiologically relevant functional information even at the single cell level: regeneration, heterogeneity in the cell population and high interference. Whole cell-based biosensors can detect a wider range of substances and can operate over a broader range of conditions such as various temperature and pH values. They present good sensitivity and selectivity and their capability for high-throughput in situ detection, however less than enzymatic biosensors. Used for water quality monitoring and toxicity assessment, drug detection and study of derived effects. Tye study of basic cellular functions and disease pathogenesis… The main issues to take care about are: (1) the selection of the reporter gene, (2) the selectivity and sensitivity of the molecular recognition that occurs when regulator proteins bind to their target analytes, (3) limitation is the diffusion of substrates and products through the cell membrane (these biosensors are slower than enzymatic ones), (4) degradability and insulating nature of cell culture matrices, (5) genetic instability in cell lines, (6) high costs of instrumentation. Living cells behave as elastic masses on the QCM (quartz crystal microbalance) surface, which implies the QCM can provide information about mass changes, reactions, and conditions at the liquid− solid interface. Cell responses can be detected by monitoring the shifts in QCM oscillation frequency and energy dissipation on the basis of the mass changes and viscoelastic properties of the QCM surface. The QCM has a wide detection range from a monolayer of small molecules to greater masses, even complex arrays of cells. Some properties of cultured cells (e.g., cell attachment, proliferation, and cell− substrate interaction) can be monitorized, even in real time. 10  Ex: Piezoelectric whole-cell biosensor with living endothelial cells (Ecs). A.3) Tissue and organelles: As whole cells, some tissues contain enzymes or enzymatic systems. Organelles do contain enzymatic systems as well, although not all that correspond to whole cells. However toxic agents, heavy metals or some detergents inhibit their enzymatic processes… They are low cost and do not require a lot of prior preparation (there is no need to purify or stabilize the enzymes and do not need of any other cofactor for enzymatic regeneration). They are also stable. Ex: Tissue-modified oxygen electrode. - It enables H2O2 detection due to catalase activity in the tissue. - Antioxidant effect is monitored by the reaction of H2O2 with tested drugs. - Measuring the inhibition of the O2 produced by catalase in the presence of H2O2. - The biosensor can be used to study the antioxidant effect of orally delivered drugs. B) Bioreceptors – Affinity Sensors: There is an interaction between the analyte and the bioreceptor (biorecognition element) without catalytical transformation. The interaction modifies an equilibrium in which is formed an analyte-receptor complex. Due to the fact that during the interaction no substrates are consumed, and no products are generated, in these biosensors, is usual to label the receptor, or an element that competes with the analyte for the union with the receptor. Labelling with fluorescent compounds, nanoparticles or enzymes. They require additional cleaning and separation steps to remove the extra labeled molecules. If enzymes are used, they require adding of specific substrates. Mass changes and optical changes in an electromagnetic radiation, are the methods most used to quantify the interaction. 11 B.1) Antibodies: The most used in bioaffinity sensors. They constitute a specific group called immunosensors. An antibody is a protein that binds selectively with an specific antigen. They algo bind with little molecules knowns as haptens (they elicit an immune response only when attached to a large carrier, as a protein). The antigens and haptens are the analytes of these sensors. Each antigen or hapten require the presence of a particular antibody. The regeneration of the immunosensor is the limiting step of these biosensors. In the actual biosensors is needed a complex antigen-antibody of high affinity, an its dissociation is usually very complicated. This limits the use of this type of biosensor to one use. It is also crucial to minimize the non-specific interaction that drive to false positives (steps of purification, isolation, etc.) – Several steps in the fabrication procedure. Maximum attention goes to the immunosensors for detection of important cancer biomarkers, such as carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA). - CEA  is a multi-tumor marker for clinical diagnosis of different kinds of cancer, such as colon tumors, breast tumors, ovarian carcinoma, gastric, pancreatic and lung carcinomas. - PSA  is a specific biomarker only for detection of prostate cancer. When specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 µg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml. Polyclonal anti-PSA antibody is used as a ―ligand‖ covalently linked to the cantilever surface. The cantilever deflection due to specific PSA binding with this antibody allows to detect PSA concentrations from 0.2 ng/ml to 60 µg/ml, which includes the clinically relevant diagnostic PSA concentration range. Human serum proteins such as HP and HSA or nonhuman serum protein such as bovine serum albumin (BSA) are used to simulate background ―noise‖, which were present at concentrations as high as 1 mg/ml. 12  Relation through the Stoney‘s Formula: Δh = 3σ(1 – v)/E*(L7d)^2. Comments of the graph: Results and Sensitivity  (1) Different cantilever geometries offer varying sensitivities. The largest deflections (indicating higher sensitivity) occur with the longer and thinner cantilevers. (2) In terms of sensitivity, the curves show that PSA concentrations can be detected across a very wide range (from 0.2 ng/ml to 60 µg/ml). (3) The line representing the clinically relevant concentration of fPSA crosses one of the curves, suggesting that the technique is sensitive enough to detect PSA at clinically significant concentrations, as indicated in the abstract. Conclusions  The graph demonstrates that microcantilevers, when designed with specific geometries, are highly sensitive for detecting very low concentrations of PSA in complex media (such as with albumin), indicating their potential utility as a diagnostic tool in clinical settings. Additionally, sensitivity varies with the cantilever's geometry, with the longer and thinner cantilevers being the most sensitive to small PSA concentrations. Antibodies + Labels: Many of the current immunosensors are variations of enzyme-linked immunosorbent assays (ELISA), differing in detection by virtue of either enzymatic, fluorescent, or chemiluminescent labels, which report on the specific formation of the immune complex (i. e. PSA). **Image: Bioconjugates for signal amplification strategies in electrochemical immunosensors  After the capture antibody is immobilized on the sensor surface, and the analyte protein is captured, these bioconjugates bind with analyte in a sandwich immunoassay. An electrochemical signal is generated using a substrate suitable for the electroactive species (typically enzyme) on the bioconjugate probe. I.e. Two distinct monoclonal antibodies directed against different PSA (Prostate-specific antigen) epitopes are used in a ―sandwich assay‖ format for capture and detection, which enhances the specificity. 13 Antibodies + Enzymes: We can use electrochemical transductors. An enzyme with a secondary intention is needed. **Image: Scheme of the sandwich assay for protein biomarker detection using magnetic beads  Antibody-modified magnetic beads capture the protein from sample solution, followed by binding of a second enzyme-labeled antibody. Enzyme‘s substrate is used to develop the electrochemical signal. WE is the working electrode. Magnetic NPs are used for solution-based capture of analytes and then drawn to the surface using an external magnet for electrochemical detection. PSA was captured on the magnetic NPs, and the captured NPs were bathed with AP-enzyme-labeled antibody and measured using differential pulse voltametry, achieving a detection limit of 1.4 ng mL1 for the PSA. Magnetic beads have fast reaction kinetics compared to bulk solid surfaces, high surface area per unit volume (owing to their small diameter), and good stability. And a relative ease of surface modification with functional groups, DNA, enzymes, or antibodies. Nucleic Acids: Nucleic acids are molecular structures formed by repetition of a unit molecule that is the nucleotide. The nucleotides are the basic structural units that constitute the chains of DNA. It is relatively easy to synthetically construct chained sequences of nucleotides that respond to the structure of certain previously identified genes. These chains will bind specifically to their complementary sequence by hybridization. In biosensors for DNA analysis (also known as genosensors), this process is used in the stage of biorecognition. Its use extends both to the processes of genetic sequencing and gene expression analysis, such as detection of mutations and DNA alterations associated with certain diseases. DNA sensors are usually labelled with fluorescent markers, although numerous detection methodologies coupled to various detection devices transduction. Ex: Detection of specific DNA sequence of the hepatitis B virus. (1) Immobilization of oligonucleotide probes on graphite electrode (poly(GA) or HepB1). (2) Oligonucleotide targets were applied (poly(CT) or HepB2, respectively). (3) For detection, ethidium bromide (2.0 × 10−2 mol L−1, 15 μL) was added to electrode surface. 14  Differential pulse voltammograms of graphite electrode containing poly(GA): before hybridization (a), and after 15 min of incubation with complementary target poly(CT) (b), or non- complementary poly(GA) (c). Electrolyte: phosphate buffer (0.10 mol L−1), pH 7.4. Modulation amplitude : 0.05 mV. Pulse interval: 0.2 s; 5 mV s−1. B.2) Aptamers: An aptamer is a short sequence of artificial DNA, RNA, XNA or peptide: a sequence of oligonucleotides (DNA or RNA) of single chain, artificially synthesized capable of recognizing various target molecules with high affinity and specificity. They resemble antibodies and are sometimes classified as chemical antibodies. Aptamers and antibodies can be used in many of the same applications, but the nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. This difference can make aptamers a better choice than antibodies for some purposes. The aptamers are obtained through a process called “Systematic evolution of ligands by exponential enrichment” (SELEX), also referred to as in vitro selection or in vitro evolution. They fold in space and acquire a conformation with certain regions to which the analyte can bind. Their principal drawback is the complexity of the obtention and the fact that its structure must be well-defined. There are numerous applications of these molecules associated with biosensors: therefore, they are named with the term aptasensor. SELEX: It is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. The process begins with the synthesis of a very large oligonucleotide library, consisting of randomly generated sequences of fixed length in which a small number of random regions are expected to bind specifically to the chosen target. The sequences in the library are exposed to the target ligand- which may be a protein or a small organic compound- and those that do not bind the target are removed. Then elution is produced to separate the target molecule and several rounds are performed. Ex: Progesterone is a small steroid biomolecule. The cyclical change in its expression during the reproductive cycle plays an essential role in the development and maturation of oocytes. An aberrant level of progesterone in women is predictive of unsuccessful pregnancy associated with abnormal uterine bleeding, early embryonic mortality, ectopic pregnancy, and so forth. A high level of progesterone has been reported in water bodies polluted by 15 industrial effluents. Consumption of such water by human, animal, and aquatic life leads to several reproductive complications, including infertility as it acts as an endocrine disruptor. ✓ Electrochemical impedance spectroscopy (EIS) was performed using a potentiostat three electrode system to develop a progesterone aptasensor. ✓ Buffer: The 5 mM K3Fe(CN)6 and 25 mM 6KNO3 electrolytic solution was applied as redox reporters to measure the electrochemical signal at the electrode surface. For EIS, each measurement was swept from 10 kHz to 0.1 Hz at a bias potential of 0.3 V with an alternating potential of 10 Mv. **Oligonucleotides carry a negative charge; the net charge of the solution will change if they bind with something that neutralizes their charge, and this difference in charge will be noticeable. **Example results show the imaginary Z plotted against the real Z, with an increase in current as concentration rises  Why?  The surface of a material will have a resistance, and when you add aptamers and the analyte, the impedance changes. If you increase negative charges, impedance, current, and concentration all increase. B.3) Lectins: Lectins are a group of non-immune glycoproteins that share the property of binding specifically and reversibly to carbohydrates, either free or as part of more complex structures. Lectins form conjugated structures such as lectin-glycoprotein, lectin-enzyme, and lectin-antibody. As recognition molecules, they are easily usable, cost-effective, and widely available and can be associated with different types of transducers, especially mass sensors. Are inexpensive compared to popular antibodies and nucleic acid. Antibodies, despite their unique specificity and selectivity, are less stable and expensive. The nucleic acid based probes such as aptamers also suffer from long, labor-intensive, and costly production processes. Moreover, the target structure of antibodies and aptamers must be well-defined. Otherwise, the cross-reactivity probability increases and undermines the accuracy of the results. They have a smaller size that can provide an appropriate surface density. Their major drawback is that they have less specificity than antibodies. 16 Images: Electrochemical detection of carbohydrates is classically based on redox reactions involving glycoenzymes, especially glucose oxidase (GOx). Left leyend: (1) lectin; (2) standard glycan core; (3) antibody; (4) CH moieties; (5) electrochemical transducer. Right image transducers: Electrochemical (A), Optical Fluorimeter (B) and Optical (C). I.e: Porous silicon (PSi) has unique optical and chemical properties which makes it a good candidate for biosensing applications. Lectin-conjugated PSi-based biosensor for label-free and real-time detection of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) by reflectometric interference Fourier transform spectroscopy (RIFTS). B.4) Receptors and Ionic Channels: Receptors are cellular regions that allow the interaction of certain substances with the mechanisms of cellular metabolism. They are proteins or glycoproteins present in the cell membrane, organelle membranes, or the cellular cytosol, to which other chemical substances, called signaling molecules, such as hormones and neurotransmitters, specifically bind. Particular interest  transport proteins, such as ion channels, as they enable the control of the flow of their associated ions (shape and charge) and monitoring through miniaturized electrochemical methods. When the target analyte binds to the ion channel or triggers a reaction with the receptor, it can lead to changes in ion flow across the membrane. These changes in ion flow can be detected and measured, providing a readout of the analyte concentration. Ion channel based biosensors are often used in electrophysiological studies or for monitoring ion specific activities. 17 **Image: Schematics of different membrane architectures implemented for biosensing: (Ai) Hybrid bilayer consisting of a self-assembled monolayer and a lipid monolayer which can be self- assembled; (Aii) A solid-supported lipid bilayer suitable for cantilever (acoustic), waveguide and silicon nanowire sensors; (Aiii) A tethered supported lipid bilayer with spacers suitable for plasmonic and impedance measurements; (B) A hydrogel-cushioned lipid bilayer decoupled from the solid support; (C) A miniaturized cushioned lipid bilayer gating a field-effect transistor (so-called BIO-FET); (D) Nanopore- spanning lipid bilayer for nanoscale optical and electrochemical sensing. **Image: Cantilever makes a direct measure, mechanical. If the biosensor is yellow, is probably made of gold, which means it has plasmons. **Image: Schematic representation of biosensor based on bacteria (which is the bioreceptor) respiring and thus reducing an electron acceptor such as NO2− to a gas (in this case N2O) that can be detected by an amperometric Clark-type gas sensor. Such sensors have been realized fore NO2−, NOx− (i.e., NO3− + NO2−) and for SO42− that is reduced to H2S. 18 B.5) Molecularly imprinted polymers: Molecularly imprinted polymers, or PIMs, are synthetic matrices derived from materials capable of adopting the shape of the molecules present in the environment in which they polymerize and persist within the material as molecular templates. Someconsider them as"artificialantibodies" (Plastibodies). In theory, they possess the ability to selectively recognize and interact with the molecules that left the corresponding imprint on their structure. Thus, they are biomimetic materials that replicate the recognition mechanism of conventional biochemical systems, as previously mentioned (enzyme-substrate, antigen- antibody, etc.). Among their advantages, they are generally believed to offer greater stability compared to other biological recognition systems. These polymers exhibit a range of highly interesting characteristics for their use as biosensors, particularly in the field of pharmaceutical and environmental analysis, due to their potential to develop PIMs aimed at a wide variety of analytes for which biological recognition systems are not yet available. They represent a relatively recent field of research, so their applications will increase in the future, especially when biologically active artificial derivatives are obtained, and when the development of competitive inhibitors, agonists, or ligands is investigated. **Image: Non covalent imprinting  Uses weaker, reversible interactions (non-covalent) between the template and monomers, leading to a flexible and widely used approach. Steps: (1) Self-Assembly  The template molecule (represented by the grey sphere) is mixed with functional monomers. These monomers interact with the template through non-covalent interactions such as hydrogen bonding, electrostatic forces, or van der Waals forces. (2) Polymerization  After the monomers have self-assembled around the template, polymerization occurs, creating a cross-linked polymer matrix (blue structure) around the template. This matrix has cavities that conform to the shape of the template molecule. (3) Template Removal  The template is removed, leaving behind cavities or binding sites within the polymer that are complementary in shape and size to the template molecule. (4) Rebinding  The polymer is now able to selectively rebind to the original template or structurally similar molecules because the cavities formed are tailored to the template. Covalent imprinting  Involves the formation and subsequent cleavage of covalent bonds between the template and monomers, offering more specific imprinting but requiring more complex chemistry for template removal. Steps: (1) Reversible Covalent Linkage  In this approach, the template forms covalent bonds with the functional monomers, creating a more specific and controlled interaction between the template and the monomers. (2) Polymerization  Similar to the non-covalent method, polymerization occurs around the template, forming a cross-linked polymer matrix. (3) Template Removal  After polymerization, the covalent bonds are reversibly cleaved to remove the template, leaving behind a cavity that mirrors the template's shape and functional groups. (4) Rebinding  The resulting polymer is capable of selectively rebinding the template with high specificity due to the precise nature of the covalent interactions during the imprinting process. 19 Types of MIPs: - Acrylate-Based Polymers: Methacrylates, acrylamides, and acrylic acid. - Polyvinyl Polymers: Polyvinyl alcohol (PVA). - Silicone-Based Polymers: Silicone polymers, like polydimethylsiloxane (PDMS). - Polystyrene-Based Polymers. - Polyethylene Glycol (PEG). - Nylon Polymers. - Polyurethane. - Hydrogels. - Co-Polymers. Flexibility and ―ease‖ of synthesis, they do not require refrigerated storage, and they work in more extreme physiological conditions of temperature and pH. Re-usable andrelatively low-cost. In general, MIP fabrication includes the preparation of the monomer-template complex before the polymerization, which requires optimization of the ratio of the template and monomer. The process of template extraction can be made chemically or electrochemically. It can be relatively time intensive. Inefficient methods can result in the collapse or rupture of the cavity after the template has been extracted. Improper template extraction can also lead to the creation of distorted binding points creating poor selectivity and thus significantly impacting the performance of MIP-based sensors. MIPs also encounter challenges with selectivity arising from a relatively poor understanding of the protein- MIP interactions. In the case of electrochemical biosensors, one significant advantage of MIPs over other bio recognition elements is the ability to fabricate MIPs directly on the electrode surface. Integration of the analyte in the MIP: A) The covalent imprinting approach relies on the formation of covalent bonds between the template and the functional monomer before the polymerization. B) The non-covalent imprinting approach, non-covalent interactions such as Van der Waals forces, hydrogen bonding, π-π interactions, dipole-dipole interaction, and ion-dipole interactions take place between the template and functional monomer. The extraction of the template is relatively easier in this method compared to the covalent imprinting approach. C) The semi-covalent imprinting approach is a hybrid: the target cavity is created using the covalent imprinting approach. However, the re-binding of the target with the created imprint happens due to non- covalent interactions. This method is widely suited for a range of polymerization conditions. D) The metal-mediated imprinting approach utilizes metal ions either as templates or as part of the template-functional monomer interactions. The strength of the interactions can be tuned based on the oxidation state of the metal ions. 20 Extraction of the template: A) The template-to-functional monomer ratio is crucial to ensure optimal creation of the cavities in the MIP. Generally, the functional monomer is always taken in molar excess as compared to the template molecules. B) The number of cavities created after template removal governs the available binding sites for sensing performance. The shape of the obtained cavity governs the specificity of the sensor. C) This cavity creation can be achieved by employing: - Chemicals such as acids, surfactants, or organic solvents to elute templates from the polymer matrix with high template removal efficiency. The chemicals used in these cases may work by breaking the bond between the template and polymer or by degrading the template itself so that it comes out during the washing steps. - Electrochemical methods: subjecting an electropolymerized surface to a voltage in order to change the surface properties, which causes the elution of the template (pyrrole-based MIPs) Or electrochemical-based template removal through the degradation of the embedded template. This is accomplished by applying an over potential to the trapped molecule either in acidic or alkaline electrolyte. To ascertain the optimum ratios, removal of template, shapes, etc, various characterization methods are applied such as UV–Vis, FTIR, NMR; Raman and fluorescence spectroscopy, SEM, TEM, etc. I.e. A surfactant-mediated sol-gel method for the preparation of molecularly imprinted polymers and its application in a biomimetic immunoassay for the detection of protein. Summary: Analytes Receptor or chemical Measurement technique or recognition system transduction mode Ions Enzymes and biological Potentiometric, voltammetric. ionophores. Dissolved gases, vapours, Enzymes, antibody and receptor. Amperometric, potentiometric, odours impedance, piezoelectric or optical. Substrates Enzymes, whole cells, membrane Amperometric, potentiometric, receptors, plants or animal tissue. piezoelectric, optical, calorimetric. Antibody/antigen Antigen or antibody: Amperometric, potetiometric or oligonucleotide duplex, aptamer, impedimetric, piezoelectric, enzyme labelled. optical and surface plasmon resonance. Various proteins and low Specific ligands, protein receptors Amperometric, potetiometric or molecular weight substrates, and channels, enzyme labelled. impedimetric, piezoelectric, ions optical and surface plasmon resonance. 21  Unit 3: Biosensors Surface. Part II. Immobilization Techniques: Immobilization in biosensors refers to the process of securely attaching or fixing a biological component, such as enzymes, antibodies, or cells, to the surface of the sensor. This immobilization is a crucial step in biosensor development. The biological component of any biosensor must be incorporated into the device in a way that ensures its activity if it possesses catalytic properties, or that the binding site where interaction with the analyte occurs is accessible to it. The choice of immobilization method can significantly impact the performance of the biosensor, including its sensitivity, specificity, and stability. Various alternatives can be employed, ranging from simple physical adsorption to the sensor surface, covalent binding, entrapment within polymeric matrices, indirect coupling through intermediate biomolecular species… A) Physical adsorption; B) Chelation; C) Disulfide Bonding; D) Ionic Binding; E) Affinity Binding; F) Covalent Binding; G) Crosslinking; H) Entrapment; I) Encapsulation. A) Physical Adsorption: Adsorption is the oldest and the simplest of all techniques. Principle: This involves adhering of the bioreceptor to the surface of the carrier via several weak non covalent interactions such as Van Der Waal’s interactions, hydrogen bond or hydrophobic interactions. This method relies on weak, reversible forces for attachment. Based in forces/bonds. Van der Waals force is a distance-dependent interaction between atoms or molecules  Lennard-Jones potential (graph: two interacting particles repel each other at very close distance, attract each other at moderate distance, and eventually stop interacting at infinite distance). 22 Hydrogen bonding: it results from the attractive force between a hydrogen atom covalently bonded to a very electronegative atom such as a N, O, or F atom and another very electronegative atom. Hydrophobic interactions: Arise from the exclusion of water molecules at the interface between two non- polar molecular areas, thereby generating a force of aggregation between these non polar areas. Carbon based materials prepared at high T are hydrophobic. Ex: Hydrogels. Advantages: Cheap, easy to perform and allows easy recovery of the bioreceptor from the carrier, thus allowing re-use of both. A variety of organic and inorganic materials can be used as support. Disadvantages: Significant bioreceptor loss cannot be avoided in this technique as the binding forces are weak, reversible and susceptible to physical parameters such as pH and temperature. The bioreceptor can be desorbed from the sensor's surface under certain conditions, potentially affecting the biosensor's performance over time. Supports used: Metallic surfaces, Metallic oxidized surfaces, Graphene, NTCs, Nanoparticles, Polymers, collagen and starch. Enzymes: Metallic surface (as Au), polymers, Reduced Graphene Oxide (hydrophobic) Antibodies: Metallic oxidized (as Titanium Oxide, Silica, Alumina...) surfaces and graphene/carbon NTCs Nucleid Acids: Au surfaces or Au Nanoparticles. In general, due to the nature of the union achieved through adsorption, both the spatial orientation of immobilized molecules and the accessibility of analytes and substrates to their binding sites are subject to chance. However, it is a popular methodology. B) Chelation: Chelation is a type of bonding of ions and their molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These ligands are called chelants, chelators, chelating agents, or sequestering agents. Chelation is capture of positively-charged metal ions by a large molecule. Example: EDTA (EthyleneDiamineTetraacetic Acid). EDTA has the capacity to chelate almost every positive ion in the periodic table (EDTA to liberate it with metal ions to the media). 23 *Oxide surface, oxygen with an empty ligand, so we can join metallic atoms, (if it is alumina we join the EDTA which increases the affinity), if it is for metallic atoms it is directly. In biosensors: Principle: It is based on the ability of charged/polar molecules, as amino acids (histidine, lysine, phenylalanine, cysteine and tyrosine), to bind to metal ions via coordinate bonds. The metal ions bound to the support surface, have metal ligands weakly bound to them. Upon exposure of the bioreceptor to the system, the weak ligands are replaced by the these. Is a reversible method (introducing a ligand with greater affinity for the metal ion). However, it is expensive and may involve safety issues to take care about. This method is less popular in industries. *Analite: Peptide. **Bioreceptor: the one that is attached to the metal (green). *** From step 2 to 3: Step after the analite binds to the bioreceptor: u can reuse the bioreceptor. ****Elution/separation: also used in aptamers, be able to separate the bioreceptor from the metal ions, u can do it by changes on the ph, imidazole, compit with the bioreceptor for the blue ligand, an this has more charge, so its going to win an push it, and removing the bioreceptor and the metal goes to the media through edta. I.e. Bonding of metal ions to the peripheral amino acids of the enzyme: Lipase immobilization could be accomplished by ionic interactions between the negatively charged side chains of amino acids of the enzyme and the protonated amino groups present in MCM-41@PEI. Functionalised with NH cause metal really like this join. 1) MCM-41@PEI-M (metal ion); 2) MCM-41@PEI-M-Lipase (enzyme + metal ion); 3) Analite: Lipds. 4) Enzyme: Electrochemical transduction, but optical in case you use a fluorofore. 24 The highest biocatalytic activity at extremely acidic and basic pH (pH = 3 and 10) values were achieved for MCM-PEI-Co and MCM-PEI-Cu, respectively. C) Disulfide Bonding: Acompound containing a R−S−S−R′ functional group. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. Bioreceptors like antibodies, enzymes, peptides, aptamers, and proteins with accessible thiol groups (cysteines) are suitable for immobilization through disulfide bonding, particularly on gold surfaces or substrates that are thiol reactive. Or modified surface to present thiols. 1) Thiols like SH (gold); 2) One thing is how you stick the bioreceptor (through disulfide bonding) and other what does my surface need to stick in it the system (for example free NHs). Proteins stick to de disulfide. D) Ionic Bonding: Ionic bonding relies on the electrostatic attraction between oppositely charged molecules or functional groups, making it useful for immobilizing bioreceptors with charged groups onto surfaces with opposite charges. Charged bioreceptor and charged surface with the opposite. Bioreceptors like proteins, enzymes, DNA/RNA, aptamers, polysaccharides, and peptides that carry charged groups can be immobilized through ionic bonding on surfaces with opposite charges. The pH of the environment, the isoelectric point of the bioreceptor, and the surface charge of the substrate are key factors in determining the effectiveness of ionic bonding. (Gold is charged with plasma, polymers extremes can be charged, etc). Supports: Polymers, Metal Oxides, Au, CNTs, GO, ITO (oxido de indio titanio, very good in chemical and electrical properties), Hydrogels… 25 E) Affinity Binding: Principle: This method is based on the specific and reversible interaction between a bioreceptor and an anchor molecule (ligand) present on the support. This process takes advantage of pre-existing biological affinities such as antigen-antibody, biotin-streptavidin or lectin-sugar interactions. These interactions are strong, specific and selective, which ensures oriented binding of the bioreceptor to the support. Advantages: Provides specific and targeted immobilization, increasing bioreceptor efficiency by effectively exposing active sites. Denaturation of the bioreceptors is avoided, since the forces involved do not affect the native structure of the biomolecule. Compatible with a wide variety of bioreceptors. Disadvantages: The process can be more expensive due to the need for specific ligands. Sensor regeneration can be complicated, as reversible desorption of the bioreceptor may not be trivial in certain systems. Preparation of the ligands may also require complex additional steps. Supports used: Polymers functionalized with ligands, functionalized nanoparticles, surfaces with antibodies or aptamers. F) Covalent Binding: Principle: In this method, the bioreceptor is permanently attached to the support through covalent bonds. This is achieved by the reaction of functional groups on the surface of the support (such as amine, carboxyl, or thiol groups) with complementary reactive groups on the biomolecule. Unlike physical adsorption, covalent bonds are strong and stable, ensuring long-lasting immobilization. Advantages: Provides a robust and stable bond, which improves the life and stability of the biosensor. Allows the use of a wide variety of bioreceptors. The resistance to adverse conditions such as changes in pH or temperature is significantly greater than in physical adsorption. Disadvantages: The preparation of the support can be complex and may require specialized chemistry for the activation of functional groups. There is a risk of denaturation of the bioreceptor during the covalent binding process, which may affect its functionality. Additionally, sensor regeneration is difficult due to the irreversible nature of the bonds. Supports used: Functionalized polymer surfaces, nanoparticles with reactive groups, materials with chemically modified surfaces (e.g., gold, silica, activated polymers). G) Crosslinking: Principle: In this method, the bioreceptors are linked to each other or to the support using crosslinkers that form covalent bonds between the biomolecules. Cross-linking agents typically have two or more reactive groups that react with functional groups on biomolecules and support surfaces. The most common crosslinkers include glutaraldehyde and carbodiimides. Advantages: Allows the immobilization of multiple bioreceptors, improving the stability and resistance of the system. It is an effective method for the immobilization of enzymes and proteins. The formation of cross-linking networks can increase the structural stability of bioreceptors. Disadvantages: Cross-linking can denature biomolecules if not properly controlled, which can affect their functionality. The process can result in limited access of the analytes to the active site due to the network formed by the cross-linking agent. It is difficult to regenerate once the bioreceptor has been immobilized. Supports used: Polymers, nanoparticles, surfaces functionalized with reactive groups (amine, carboxyl). H) Entrapment: Principle: In this method, bioreceptors are physically trapped within a polymeric matrix or gel without forming direct bonds with the surface of the support. The trapped molecules are confined within the matrix, but can still interact with analytes in solution. The matrices used can be hydrogels, membranes or thin films. 26  Advantages: Provides good stability to the bioreceptor without the need to chemically modify its structure. The process is gentle and does not affect the biological activity of the immobilized molecules. Matrices can protect bioreceptors from external factors, such as pH or temperature variations. Disadvantages: Diffusion of analytes through the matrix can be slow, which could reduce the response speed of the sensor. There is a risk that the bioreceptor will become trapped in an orientation that makes it difficult to access the analyte. Regeneration can be difficult due to the nature of the trapping matrix. Supports used: Hydrogels, membranes, polymer films, sol-gels. I) Encapsulation: Principle: This method consists of encapsulating the bioreceptor within a micro or nanostructure such as liposomes, nanoparticles, or microspheres. Encapsulation keeps the bioreceptor protected in a controlled environment, allowing it to interact with the external environment through pores or semipermeable membranes. Advantages: Effectively protects bioreceptors from adverse environmental conditions and degrading agents. It allows a controlled release of the bioreceptor or biological products. It is ideal for the immobilization of sensitive bioreceptors or those that require a specific environment to maintain their activity. Disadvantages: The encapsulation process can be complex and expensive. Diffusion of analytes or products through encapsulating membranes can limit the response speed of the sensor. Controlling the size of encapsulating structures is crucial for optimal functionality. Supports used: Liposomes, nanoparticles, polymer microspheres, semipermeable membranes. Unit 4: Fabrication and Materials. Surface Functionalization: Modification of the surface of the materials, in general by functional groups, in order to modify the properties of the material:  Physical functionalization.  Chemical functionalization.  Tribological functionalization.  Biological functionalization (biofunctionalization). 1) Physical Functionalization: Functionalization based on the use of physical techniques (thin film deposition techniques). Special interest in: a) The interface material/coating. B) Surface cleaning and preparation to improve adhesion (chemical methods, plasma, etc.) 2) Chemical Functionalization: Use of chemical and dissolution techniques, to also protect the surface. Focused on modifying (optimizing) chemical properties (chemical stability, corrosion, photodegradation). Ex. Au or Pt coating. 3) Tribological Functionalization: Modification of the materials in order to improve their tribological properties (friction, wear and lubrication during the contact in movement of solid surfaces). Ex. Plasma.  Bending, Twisting, etc. 27  4) Biological Functionalization - Biofunctionalization: Fixation on the surfaces of functional groups for the immobilization of biomolecules (proteins, nucleicacids)  Immobilized functional groups in general.  Reactive functional groups I.e. Covalent binding… Hidroxyl: R-OH. Suldhydril (Thiol): R-SH. Carboxyl: R-COOH. Amine: R-NH2.  Native biomolecules  They must keep their biological functionality. Physical/Chemical Functionalization: Thermal Evaporation: The source material (precursor) is heated to high temperatures until it reaches a vapor pressure high enough that a significant fraction of atoms or molecules on the surface transitions to the vapor state. Low Vacuum: 10^-2 – 10^-5 mbar. Metals: Au, Ag, Pt, etc. Low-density coatings (sometimes with poor adhesion to the surface). Sputtering: Extraction of atoms from the source by intense bombardment of the target material with ions generated in an electric plasma discharge. Aprox. 10^-3 mbar (deposition process) (10^-6 mbar before Argon). Metals: Au, Al, Ag, Pt, etc. Semiconductors: Si, Ge. Ceramycs: Si, C. Alloys Coatings with higher density and improved crystalline quality. PLD – Pulsed Laser Deposition: We use a laser to "remove" material from a target through various physical and chemical mechanisms known as ablation. The material evaporates from the target due to high-energy laser light radiation. High to Low Vacuum: 10^-8 a 10^-6 to >10 mbar). All type of materials. High-density coatings and improved adhesion to the substrate. MBE – Molecular Beam Epitaxy: Epitaxial deposition: The evaporated material impacts the substrate as neutral molecular or atomic beams at a controlled temperature in an ultra-high vacuum atmosphere (UHV). 10^-10 a 10^-11 mbar. Metals: Au, Al, Ag, Pt, etc. Semiconductors: Si, Ge. Alloys Oxides, etc. Deposition of a crystalline layer onto a crystalline substrate. 28  Chemical Vapor Deposition (CVD): Vapor phase technique: Reaction of one or more compounds in the gaseous form to produce a solid product. Homogeneous reaction  Reaction in the gas pase – NPs. Heterogeneous reaction  Reaction in contact with the support substrate. (Moderate vacuum) Metals (Al, Au, W…). Semiconductors (Si, Ge, GaAs, GaP…). Oxides and Silicon Nitrates. Graphene. CNTs Graphene: Good conductive properties, obtained by CVD or Exfoliation with Sticky Tape: By Exfoliation: - Mechanical cleavage using Scotch tape on bulk graphite. - Direct liquid phase exfoliation of graphite/ graphite intercalation compound with the help of ultrasonication or via oxidation of graphite to graphite oxide (GO). - Exfoliation of GO to graphene oxide by ultra sonication followed by reduction process to restore electronic properties (reduced graphene oxide-RGO). Relative high crystal quality (high electrical conductivity, less crystal defect) but the production yield is still low. Product are usually contaminated by organic impurities and its is difficult to control the number of graphene layers (whereby increasing the number of layers decrease its optical transparency). By CVD: Graphene can be produced by molecular growth from small molecular carbon precursors by Chemical Vapor Deposition (CVD) or epitaxial grown on a substrate, where well controlled thickness (number of layers) can be done using different substrate catalysts and growing parameters. Graphene by CVD method have large area, high quality and this method has the best potential for mass production of high purity graphene. 29  Sol- Gel: It is used to coat graphene with other materials (metal oxides, silica oxide, etc) or modify graphene-based surfaces. The conversion of monomers in a colloidal solution (sol), which is a precursor to an integrated network or gel. Precursors with Silicon, aluminum, titanium, zirconium... It is usually used to grow metal oxides, ceramic and vitreous materials. NPs – Coprecipitation: To obtain metallic nanoparticles. Reaction of two or more soluble precursors in an oversaturated solution results in the formation of a sparingly soluble compound (product). Principally: Metallic NPs (Au, Ag…). Quantum dots (Cd, Zn/C, Se/S, Se… Magnetic NPs (superparamagnéticas) (Fe3O4…). Electrodeposition: Gold nanoparticles: coprecipitation or chemical vapor deposition. Carbon nanotubes: chemical vapor deposition. Graphene: cvd, exfoliation. You deposit conductive materials that can be put into solution, conductive polymers, on conductive material: metal, doped semiconductor, not normal, not much conduction is necessary. The process by which metal ions present in a solution are incorporated into a substrate through a reduction reaction. Metals. Au, Pt, Fe, Ni, Cu, Bi… Allloys (FeNi…) Conductive Polymers (PEDOT…) Tribological Functionalization: Modification of the materials in order to improve their tribological properties (friction, wear and lubrication during the contact in movement of solid surfaces). Tribological properties indicate how materials perform when in contact with mating materials under dynamic conditions. Heat treatment, thermochemical treatment and vacuum deposition of lubricant thin films. Laser ablation of materials. Nanostructuration of the surfaces. 30  Lattice Dismatch: You cannot mix materials that you do not like: because they can fragment or separate. Where as and af are the lattice parameters (shortest interatomic distance) of the substrate (as) and the film (af). a) no tension, almost always, it's going to be the same material. b) in tension. c) in tension while it grows, it generates vacancies and adds extra atoms. d) it's broken, they separate. A film deposited on a substrate will be in compression if the lattice parameter of the film is greater than that of the substrate, and vice versa. It is necessary to know the orientation relationship between the film and the substrate at the interface for calculating the lattice misfit (f). What would you do if you need to have a specific bulk substrate and a specific material in the surface, and they don’t match?  Introduce a buffer or intermediate layer between the bulk substrate and the desired surface material. This buffer layer can help bridge differences in chemical properties or lattice mismatch, acting as a mediator. Cobre (Cu), Oro (Au), Paladio (Pd), Platino (Pt). Silicio (Si) y Germanio (Ge). Aluminio (Al) y Galio (Ga). Arseniuro de Galio (GaAs) y Aluminio Arseniuro (AlAs). Grafeno y Disulfuro de Molibdeno (MoS₂). Silicio Carburo (SiC) y Diamante (C). Surface Functionalization – Biofunctionalization: Biofunctionalization techniques (surface/functional group dependence): Thiol groups on Au and SiO2  SH. Hydrosilization  Alkenes and alkynes (H) (addition of Si-H). Silanization  Si + functional group (OH).  Applications: Biosensors; Tissue engineering implants and prosthesis; Drug control release.  There are more: Biotin-Streptavidin Binding, H2O binding, gels, etc… 31  Image: Illustration for the coordination bonding between a metal surface and a ligand and the basic designs for common Au/Fe3O4 nanoparticles. (AI) Interaction between a gold surface and thiol groups; (AII) Interaction between an iron surface and a dihistidine group; (AIII) Interaction between an iron surface and a catechol group; (BI) a gold-coated Fe3O4 nanoparticle; (BII) a Au/Fe3O4 Janus nanoparticle; (BIII) a gold-dotted Fe3O4 nanoparticle assembled via a thiol-functionalized core. Image: Formation and release of the zinc complex by the reaction of active hydroxyl groups (OH) on the surface oxide film with a zinc chloride reagent. Techniques of analysis and characterization of surfaces with biological systems Scanning and transmission electronmicroscopy: SEM y TEM. Atomicforcemicroscopy: AFM. Fluorescenceand confocal microscopy. Infrared spectroscopy. But not only for characterization of the surface, also as measure of a change in the properties. Unit 5: Electrochemical Biosensors. General Concept: They utilize electroanalytical properties that are produced or modified by the interaction between the analyte and the recognition system, providing quantitative or semi-quantitative analytical information. For the proper functioning of the biosensor, the recognition element and the transduction system must be in direct contact. Measuring changes in: Potentiometric – Electrode potential. Amperometric–Generated or consumed electric current. Conductimetric – Electrical conductivity. Impedimetric –Electrical resistance. Ionic charge or Field effect – Charge density in an ISFET transistor. 32 Types: The two types of electrochemical biosensors with three electrodes: reference (RE), working (WE), and counter (CE) connected to a potentiostat  3 electrode Cell. The frit (en la última imagen, parte de abajo del tubo es un ceramic frit) allows ionic transport into the electrode. In order to operate properly the frit must always be kept wet with electrolyte (is permeable to certain ions, by choosing the pore size). We need conductivity through the solution. We need that the impedance (resistance) that that current feels across the solution to be as small as possible. SPE – Screen Printed Electrode Holder: B: Summary of attachment options depending on the bioreceptor. FET is sometimes for direct measurement, without bioreceptor. Also section: Optical and Electronical lithography. What is going on without doing anything: There is a potential difference between the liquid electrolyte and the substrate, referred to as Equilibrium potential: Nerst  Any Redox processes are in equilibrium. There is no current flowing through the system. A) Impedimetric Electrochemical Biosensor: Example: This study presents a novel microfluidic aptamer-based electrochemical biosensing platform designed to monitor damage to cardiac organoids. It addresses challenges faced by traditional biosensing techniques such as limited sensitivity, selectivity, stability, and the need for large working volumes. By using aptamers that specifically bind to the creatine kinase (CK)-MB biomarker, a marker of cardiac tissue damage, this biosensor offers high sensitivity and selectivity, surpassing antibody-based sensors. The sensor‘s effectiveness was demonstrated using a heart-on-a-chip model, where human stem cell- derived cardiomyocytes were exposed to doxorubicin, a cardiotoxic drug. The biosensor successfully 33 detected trace amounts of CK-MB released by the cardiac tissue in a dose-dependent manner, correlating with the organoid's beating and viability Tioles + Aptameros (bioreceptor) + Antigenos (analyte). More about impedimetric electrochemical biosensor: In electrochemical impedance spectroscopy (EIS) used in impedimetric biosensors, a small alternating current (AC) voltage is applied (typically sinusoidal and only a few millivolts). This is crucial because it minimizes any disturbances or significant chemical reactions in the system, ensuring that the biological environment remains unaltered. Unlike other methods that might require higher voltages to trigger chemical reactions, in impedance techniques, you can obtain the impedance of the system without inducing strong electrochemical processes. Additionally, when working with a liquid-solid interface (such as in biosensors), the system can be modeled using an equivalent electrical circuit. These circuits simulate the behavior of the system based on parameters like resistance, capacitance, and other impedance-related elements. However, if high voltage amplitudes or non-linear components are introduced, the system‘s response would no longer be linear, making it difficult to simulate accurately. By maintaining low-amplitude AC signals, you ensure that the system remains in a linear, stable state, allowing for precise modeling of the biological and electrochemical interactions. This method is particularly useful for biosensing applications where sensitive detection of small biological changes is required without altering the sample. Image Potentiostat: Setup of a potentiostatic electrochemical cell used for electrochemical analysis and characterization. Potentiostat - An electronic device that controls the potential difference between the working electrode and the reference electrode  Personal Computer - Used to control the potentiostat and analyze the data obtained from the electrochemical measurements.  Working Electrode - The electrode where the electrochemical reaction of interest takes place.  Reference Electrode - Provides a stable and known reference potential against which the potential of the working electrode is measured.  Counter Electrode - Completes the electrical circuit and allows the passage of current between it and the working electrode.  Electrolyte - The solution containing the ions necessary for the electrochemical reaction. 34 The graphs on the right side of the image show typical electrochemical data obtained from this setup, such as the current-voltage (I-V) curve and the Nyquist plot, which are used to characterize the electrochemical properties and kinetics of the system. A sinusoidal voltage of low amplitude (Ej. 10 mV) and sweeping in frequency ( 0) is applied to an n-type semiconductor, it pulls electrons (negative charge carriers) towards the surface, causing the conduction band (ECE_CEC) to bend downwards, creating an accumulation of electrons near the surface. Depletion (Middle column: (b)):  p-type (top): When a small positive bias (V > 0) is applied to a p-type semiconductor, the holes are repelled from the surface, leaving behind negatively charged acceptor ions. This causes a 48  region with few mobile charge carriers (electrons or holes), called the depletion region. The energy bands bend downwards as the valence band moves away from the Fermi level.  n-type (bottom): When a small negative bias (V < 0) is applied to an n-type semiconductor, the electrons are repelled, leaving behind positively charged donor ions. This results in a depletion region at the surface, and the conduction band bends upwards away from the Fermi level. Inversion (Right column: (c)):  p-type (top): When a strong positive bias (V >> 0) is applied to a p-type semiconductor, the positive voltage pulls electrons from the bulk into the surface region, inverting the behavior. Now, the surface behaves like an n-type semiconductor (with more electrons than holes), and the conduction band (EC) drops below the Fermi level, while the valence band (EV) bends down significantly.  n-type (bottom): When a strong negative bias (V 0 (n-channel!): Since VS = VD = 0, the Fermi levels in the source, device, and drain all align; the device is in equilibrium, and nocurrent flows. A positive gate voltage increases the electrostatic channel, potential which in lowers conduction band. 49  VG>0 and VD>0 (n-channel!): VD is not 0, the Fermi levels in the source, device, and drain are not aligned. Applied to Fermi Energy: Numerical simulations are needed to resolve Fn(x) (Fermi in the n channel), but it is clear that there will be a slope to Fn(x), so current will flow. More FET information: The FET-type biosensor is one of the most attractive electrical biosensors due to its advantages of sensitive measurements, portable instrumentation, easy operation with a small sample requirements, low cost with mass production, and high speeds. In the biosensor configuration, the metal gate of a FET-type biosensor is generally replaced by a ―biofilm‖ layer material such as a receptor, enzyme, antibody, DNA, or other type of capturing molecule biologically specific for the target analyte. Or the biosensor surface can be biofunctionalized. In response to target molecules in the solution, the biomodified gate (G) surface modulates the channel conductivity of the FET, leading to a change in the drain current. So, the biomolecular binding event can create an electric field, similar to the control electric field applied to a conventional FET. The FET sensor is connected to an electronic circuit to monitor the specific conductance of this sensor surface. The binding of a charged biomolecules (such as nucleic acids and proteins) results in accumulation of carriers caused by change of electric charges on the gate terminal. In another words, once the analyte binds to the recognition element, the charge distribution at the surface changes with a corresponding change in the electrostatic surface potential of the semiconductor. This change in the surface potential of the semiconductor acts like a gate, changing the amount of current that can flow between the source and drain electrodes. This change in current (or conductance) can be measured, thus the binding of the analyte can be detected. The adsorption of molecules on the surface of the semiconducting channel either changes its local surface potential or directly dopes the channel, resulting in the change of FET conductance. In solution-gated FET biosensors, the analytes are detected in an aqueous environment. From the scheme, the semiconducting channels are immersed in a flow or sensing chamber. The source and electrodes are 50 insulated to prevent current leakage from ionic conduction using insulators. The gate electrode (Ag/AgCl or Pt) is immersed in the solution. Applying VCC (Vdd in the image)  Transistor bias: Point definition sign. Delaware job: we apply voltage in a transistor to make it always work equal and we know your behavior (any change, it is discarded which may be due to the electronic). Vdd measurement Vg measurement Vds measurement Image: Ag source-drain electrodes and a ZnO seed layer were deposited by RF sputtering on a cleaned quierez cacharte cunmigu? Si/SiO2 substrate. (a) Schematic illustration of the fabrication of the Fe₂O₃- ZnNRs FET-based non-enzymatic glucose sensor; (b) Surface FESEM; (c) Cross-sectional FESEM image; (d) EDX results of as-grown ZnNRs; (e) Low- and high-magnification FESEM images; (g) FESEM and EDX results of Fe₂O₃-ZnNRs; (h) XRD patterns and TEM images of a ZnNR and of the Fe₂O₃-ZnNRs; (i) The inset in (i) shows the HRTEM image of the Fe₂O₃-ZnNRs. Why the FE203?: Fe₂O₃ is a semiconductor with a relatively narrow band gap (~2.1 eV), which allows it to efficiently participate in electronic transitions. This property makes it useful in field-effect transistors (FETs) or other devices that rely on electronic conductivity changes, improving sensitivity in sensor applications. 51  Image left: (a) I–V response of the non-enzymatic FET-based glucose sensor in 0.1 mM PBS solution (pH = 7.4) without glucose and with 1 mM glucose; (b) Non-enzymatic I–V response of Fe₂O₃-ZnNRs FET in 0.1 mM PBS solution (pH = 7.4) with different concentrations of glucose; (c) Calibrated linear curve; (d) Selectivity test. The inset in (b) shows the overall calibrated curve with linear and nonlinear response. Image right: I–V response curves of the FET sensor measured in the presence of 4 mM glucose (a); calibrated response curve (b); mouse whole blood samples (c); and filtered serum samples (d). NW Channel: (A) A SiNW-FET is composed of a single SiNW (or a bunch of SiNWs), which is connected between a source (S) and drain (D) electrodes, laid on a Si wafer. (B) Receptor molecules, immobilized on the SiNW(s), are utilized to recognize specific targets with a SiNW-FET biosensor. When positively charged targets bind on an n-type SiNW FET, holes are accumulated in the SiNW leading to an increase in the electrical conductance. Conversely, negatively charged targets cause a depletion of charge carriers to reduce the conductance of SiNW. The resistivity and the Field Effect Transistor (FET) scheme: Resistivity scheme applies mostly to the metallic NW-based sensors– The metallic NWs act as metal resistors. Changes in conductivity. 52 FET scheme is used in semiconductor NW-based sensing devices. Depending on the polarity of the targeted species, these charged molecules will increase or decrease the signal relatively to the previously measured signal. D) Potentiometric Electrochemical Biosensor: Potentiometric biosensors are developed by combining a biorecognition element (essentially an enzyme) with a transducer that senses the variation in protons (or other ions) amount, the recorded analytical signal being logarithmically correlated with the analyte concentration. Potentiometric assays rely on recording the potential/pH variation. The electrostatic potential (volts [V]) (between the working electrode and reference electrode) is measured. Little or practically no current is involved in the measurement. The potential is proportional to the activity or relative concentration of species generated or consumed in the reaction A pH meter is one of the most commonly used applications of potentiometry. By definition, pH is related to the activity of H+ ions in the following way: The simplest transducer in the development of potentiometric biosensors is the glass pH electrode. Cremer discovered that the potential difference across a thin glass membrane is a function of pH when opposite sides of the membrane are in contact with solutions that have different concentrations of H3O+ (acid in an aqueous solution that is deprotonated). - Dependance with the ph. Ion-selective electrodes (ISEs). In addition to the glass pH electrode (H+ ions), ion-selective electrodes are available for a wide range of ions. It also is possible to construct a membrane electrode for a neutral analyte by using a chemical reaction to generate an ion (biosensor) that is monitored with an ion-selective electrode. In research. 53 Ex. of glass electrode (ph): Glucose oxidase immobilization is achieved using cellophane, nylon or nitrocellulose membranes that are subsequently fixed on the sensitive bulb of the pH electrode that senses the pH diminution, as a result of the biocatalytical reaction occurring in the enzyme layer (glucose oxidation by glucose oxidase). Ex. of virus detection (Au electrode): Virus particles are immobilized using thiol groups on a gold surface, creating a 3D molecularly imprinted structure. After removal of the virus, the surface retains cavities that selectively bind the virus, and virus detection is achieved through changes in electrochemical response measured by a potentiometer when the virus rebinds to the imprinted cavities. Image: Schematic illustration of the 3D molecular imprinting process: (a) Au-coated surface showing the underlying roughness of the unpolished Si wafer and (b) imprinting process where thiols and template molecules (target analytes; virus particles in this case) are adsorbed onto the Au surface. The thiols are permanently bound into a SAM that is crystallized around the template molecules. (c) Template molecules, which are weakly adsorbed on the Au surface, are removed by washing in a 3 M NaCl solution, leaving behind imprinted areas in the SAM with an inverse replica of the surface molecular structure of the template molecules. (d) Potentiometric analyte detection: When the analytes are readsorbed into the imprinted SAM layer, it creates a change in OCP. (e) Schematic example of the potentiometric response as a function of time when analytes are injected. Inset: The illustration of the potentiometric response occurring when the analyte comes in direct contact with the unprotected imprinted area on the Au contact. (f) Schematic example of the normalized potentiometric response as a function of the analyte concentration for sensors imprinted with different analyte concentrations. Inset: The illustration of the density of the imprinted regions corresponding to the different concentrations. Graphs: Response of potentiometric sensors of molecularly printed gold electrodes to detect SARS-CoV-2 and MERS proteins, as well as heat-treated proteins (HT-S). The response is based on the change in open circuit potential (OCP) and analyte concentration, demonstrating the specificity of the sensor. C) Amperometric Electrochemical Biosensor: The principle of an amperometric sensor is based on measuring current generated by catalytic or bioaffinity (with reaction) at the electrode surface, at a constant working potential with respect to the reference electrode. The amperometric biosensors monitorize the Faradaic currents due to electronic exchange between the biological system and the electrode maintained at the constant voltage. 54 Apart form the inherent selectivity of the biosensor the sensitivity is higher than potentiometric ones. Furthermore, some chemical reactions can be forced or strengthen due to the constant voltage applied. Example with glucose: The GOD based glucose biosensors rely on the biocatalytic reaction involving the reduction of the flavin group (flavin adenine dinucleotide, FAD) of GOD by glucose. The reduced form of the flavin group (FADH2) then reacts with molecular oxygen present in sample to regenerate the oxidized form of GOD (FAD). The hydrogen peroxide is oxidized at a platinum electrode. The number of electron transfer, at the electrode surface, is directly proportional to the number of glucose molecules present in blood. Three measurement strategies: - By measuring the oxygen consumption. - By measuring the amount of hydrogen peroxide produced by the enzyme reaction. - By using a diffusible or immobilized mediator to transfer the electrons from GOD to the electrode. 1) First generation uses oxygen as the electron doner and detects the decreased oxygen or liberated H2O2. Similar oxidases include choline oxidase, pyruvate oxidase, lactate oxidase, and glutamate oxidase. 55 2) Second generation biosensors use a mediator (ferrocene derivatives or ferrocyanide, etc.) and thus are oxygen independent. Redox mediator enhances the electron transfer between the redox center of the enzyme and the electrode surface. 3) Third generation biosensors rely on bio-electrocatalysis, where direct electron communication between the enzyme redox center and the electrode occurs. Third generation image: a) Truncation of oxidoreductase. The deletion of the haem 1c binding domain, which is not involved in the electron transfer pathway facilitates electron transfer. b) Surface modification. Deglycosylation of glucose oxidase decreases the distance between active site and electrode and facilitates electron transfer. c) Active site mutation. Genetic modification of a glucose oxidase to display a free thiol group near its active site which facilitates the sitespecific attachment of a maleimide-modified gold nanoparticle and enable direct electrical communication. Another example: Image: (a) Chronoamperometry plot showing the electric current response of different concentrations of SARS-CoV-2 protein as a function of time. The current densities were obtained by applying a fixed potential of 580 mV for 5s (Inset) of which the first 250 ms are highlighted. (b) The calibration plot obtained by plotting the current density value at 50 ms for different protein concentrations tested (blue points). Electrochemical Biosensors until now: Schematic diagram of (a) amperometric/voltammetric, (b) potentiometric, (c) conductometric biosensors, and (d) impedimetric biosensor with the relative equivalent circuit [(Cdl = double-layer capacitance of the 56  electrodes, Rsol = resistance of the solution, Cde = capacitance of the electrode, Zcell = impedance introduced by the bound nanoparticles, and Rcell and Ccell are the resistance and capacitance in parallel). Unit 6: Lockin Amplifiers. Synchronous Demodulation: A Lock-In amplifier is a type of amplifier that allows detect extremely weak signals in the presence of noise. It works based on a detection technique called detection synchronous or synchronous demodulation, which allows identifying and amplify specific signals that have a frequency known, even when they are buried in noise. For this uses a reference signal that is in phase and frequency with the signal of interest. Let a signal of interest be 𝑆(𝑡) = 𝐴⋅sin(𝜔𝑡), where A is the amplitude and ω the angular frequency of the signal. Let also be a reference signal 𝑅(𝑡) = sin(𝜔𝑡+𝜙), where ϕ is the phase. The output of the multiplier is the product of both signals: 𝑆(𝑡)⋅𝑅(𝑡) = 𝐴⋅sin(𝜔𝑡)⋅sin(𝜔𝑡+𝜙) = 2𝐴[cos( 𝜙)−cos(2𝜔𝑡+𝜙)]. Synchronous Detection: The output now has two components: A low frequency signal constant, that is, not oscillatory, cos(𝜙), which represents the signal interest. A high frequency signal at 2ω, which corresponds to components not desired. cos(𝜙) reflects the phase correlation between the signal and the reference; measures how much of the signal of interest is in phase with the reference. If the entry and reference are perfectly in phase (𝜙 = 0), then cos(𝜙) = 1, and we obtain the maximum value of the amplitude of the signal of interest. If they are completely out of date (𝜙 = 90), then cos(𝜙) = 0, and there is no low frequency component; the Lock-In does not detect any useful signals. The Lock-In amplifier takes advantage of this to extract a signal that is proportional to the amplitude of the signal interest and its phase alignment with the reference, Steps: 1) SIGNAL REFERENCE  The first step is to have a signal reference to the same frequency as the signal to be measured. This reference can come from a function generator or source that is already synchronized with the system. For example, if we want to detect a 1 kHz signal in presence of noise, the reference it will also be at 1 kHz. 2) SYNCHRONOUS DEMODULATION  The Lock-In amplifier uses the reference to perform a multiplication of the input signal with the reference signal, which generates two components: one of low frequency (the signal of interest) and another of high frequency (noise outside the band of interest). 57  3) LOW-PASS FILTER  The multiplied signal passes through a low-pass filter that removes the high- frequency components (noise), leaving only the signal of interest. The Multiplier : The logarithmic multiplication rule states that: Then, if we use an adder to sum the logarithms of two signals, we would achieve multiplication after applying an exponential. We will use operational amplifiers to build each of the components. 1) Logarithmic Amplifier: Shockley Equation:  𝐼: Current flowing through a diode.  𝐼𝑠: Reverse saturation current of the diode, a small current that depends on the material and temperature.  𝑉: Voltage applied across the diode (from anode to cathode).  𝑛: Ideality factor (or emission factor), which varies between 1 and 2 depending on the diode.  𝑉𝑇: Thermal voltaje. 2) Summing Amplifier: The outputs of the two logarithmic amplifiers are connected to an inverting summing amplifier. If we set R1 = R2 = Rf = R, then no amplification occurs. 58 If we connect it to the previous stage: 3) Exponential Amplifier: To reverse the logarithmic operation, we apply the inverse operation, an exponential. 4) Inverting Amplifier: Since the multiplication of two positive signals is giving a negative result, we add an inverting amplifier to correct the sign. Again, we set R1 = R2 or Rin = Rf. 59  5) Complete Multiplier: Unit 7: Optical Biosensors. General Concept: This method is based on optoelectronics, which is the study and application of electronic devices that interact with light and thus is usually considered a subfield of photonics. In this context, light often includes invisible forms of radiation such as gamma rays, X-rays, ultraviolet, and infrared. Optoelectronic devices are electrical-to-optical or optical-to-electrical transducers. In the most commonly used form of an optical biosensor, the transduction process induces a change in the phase, amplitude, polarization, or frequency of the input light in response to the physical or chemical change produced by the biorecognition process. The main components of an optical biosensor are light source, optical transmission medium (fiber, waveguide, etc.), immobilized biological recognition