Bien 545 - Ch 2 (1) (1) PDF

Chapter 2 Biomolecular detection assays Outline  Molecular biomarkers  Body fluids  Types of biomolecular detection assays 1 Chapter 2 Molecular biomarkers  Biomarker is defined as a biomolecule that has the ab...

Chapter 2 Biomolecular detection assays Outline  Molecular biomarkers  Body fluids  Types of biomolecular detection assays 1 Chapter 2 Molecular biomarkers  Biomarker is defined as a biomolecule that has the ability to play a prime role in different biological and pathological processes or give a specific pharmacological response to a drug. A biomarker is a short‐term biological marker that can be used as an indicator of a particular disease stage or some other physiological states of an organism. Biomarkers are generally of three types, i.e. predictive, prognostic, and diagnostic.  Predictive Biomarker Predictive biomarkers help in prediction of therapeutic response to a specific drug.  Prognostic Biomarkers Prognostic biomarkers are used to indicate the development of a disease.  Diagnostic Biomarkers A diagnostic biomarker detects or confirms the presence of a disease or condition of interest, or identifies an individual with a subtype of the disease. 2 Chapter 2 Molecular biomarkers Molecular biomarkers can be broadly in form of: 1. Macromolecules: DNA/RNA, proteins  With wide applications in cancer and viral diseases  For example, normal cells have different receptor proteins on their surface that are converted into cancer biomarkers when a normal cell is turned into a cancer cell. 2. Small molecules: products in metabolic pathways which may regulate biological processes Glucose Cortisol  For example, cortisol is frequently used as a biomarker of psychological stress  Continuous monitoring of small molecule bimarkers can serve as early warning for patients with critical or chronic conditions, such as kidney disease, liver disease, or heart disease. 3 Chapter 2 Body fluids  The molecular biomarkers can be obtained in a minimally or non-invasive way through the sampling of body fluids (a promising alternative to tissue biopsy), such as blood (gold- standard biofluid), urine, pleural effusion, sweat, cerebrospinal fluid, and saliva  Blood is a gold-standard source of biomarkers. The level of biomarkers that exist in other biofluids may relatively correlate with their levels in blood stream. Blood stream is a meaningful source of any macromolecules and small molecules biomarkers Biomarkers secreted by organs and cells immediately enter the blood stream and can be detected 4 Chapter 2 Body fluids Biomarkers enter ISF (interstitial fluid), sweat, and saliva via either transcellular or paracellular routes: Transcellular: diffusion through the plasma membrane of capillary endothelial cells or vesicular transport through the cell (transcytosis). Paracellular: diffusion and/or advective transport through the space between cells.  Therefore, there are close correlations between the level of the biomarkers in blood plasma and sweat/saliva/ISF Analyte Transport from blood stream to sweat and saliva 5 Chapter 2 Body fluids  Sweat is produced by eccrine and apocrine glands which are in the dermal level of skin and terminate in secretory canals. Sweat can flow into the skin surface and hair follicles.  Sweat is at moderate acidic to neutral pH levels, normally between 4.5 and 7.0.  Most of the sweat consists of water and electrolytes. There are also amounts of minerals, lactic acid, and urea in sweat. The mineral content includes sodium, potassium, calcium, and magnesium. Sodium and chloride are the primary electrolytes in sweat. Many other elements are also present in sweat, such as zinc, copper, iron, chromium, nickel, and lead. In addition, approximately 1% of the sweat consists of proteins such as immunoglobulins and glycoproteins and cytokines.  Sweat diagnostics in clinical testing include limited commercial application, which is for infant cystic fibrosis. This test detects chloride concentrations in the sweat. Chapter 2 Body fluids  Saliva is a clear and mild acidic (pH 6–7) mucoserous exocrine secretion. It is created from the salivary glands and from gingival crevicular fluid.  The constituents of saliva include a variety of electrolytes: sodium, potassium, calcium, magnesium, bicarbonate, and phosphates. There are also immunoglobulins, proteins, enzymes, mucins, and nitrogenous products such as urea and ammonia.  Currently, saliva is also used for the detection of viral diseases, gastric ulcers and cancers, liver disease, and tuberculosis. Saliva can be used to monitor levels of polypeptides, steroids, antibodies, alcohol, and various drugs. Growth factors (a kind of protein biomarker) can also be found in saliva and they play an important role in wound healing and oral health maintenance. The commercial market provides saliva test strips that can be used at home to detect alcohol, drugs, and nitric oxide. Most of the methods collect saliva through an absorbent pad, apply a chemical solution, and detect color change to indicate the result. Some methods commonly use point-of-care (POC) devices to screen for diseases such as HIV. 7 Chapter 2 Body fluids  Urine is a body fluid that can provide substantial information about individual health and disease. Urine analysis is a noninvasive means to evaluate the functional status of many organs, especially the kidney. It can also be used to monitor body homeostasis and some metabolic disease processes.  The urine of a normal healthy individual does not contain significant amounts of glucose, bicarbonate, or albumin, which are normally completely reabsorbed by the kidney. For example, Albumin is less than 30 mg L−1 in urine because of renal filtration. Hence, albuminuria can be used as a diagnostic index for chronic kidney disease.  Citrate is one of the crucial metabolites in the Krebs cycle for aerobic cells. Citrate levels in urine are related to several diseases, including kidney dysfunction and prostate cancer. 8 Chapter 2 Body fluids  Cerebrospinal fluid (CSF) is a clear, colorless liquid found in your brain and spinal cord.  CSF is an accessible source of brain-derived proteins, which mirror molecular changes that take place in the central nervous system (CNS). Consequently, CSF biomarkers and their use in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson disease (PD) represent an emerging area of research.  Molecular aberrations in the AD brain are reflected in the cerebrospinal fluid (CSF). The core candidate CSF biomarkers Abeta42, total tau (T-tau), and phosphorylated tau (P-tau) have been shown to have a high diagnostic performance to identify AD also in the early phase of the disease. Also, elevated level of α- synuclein has been observed in CFS of PD patients. 9 Chapter 2 Body fluids  For example, an effective management of cancer diagnosis screening by using body fluids containing molecular biomarkers (such as circulating tumor cells and EVs: extracellular vesicles). Can be achieved 10 Chapter 2 Body fluids Pretreatments required for analysis of molecular biomarkers After collection of the biofluid of interest, some pretreatment procedures depending on the application are required  Lysing In the cases we aim to investigate the presence of any viral infections, analyze human DNA or obtain the biomarkers in the secreted extracellular vesicles (EVs) The bacteria/cell/virus/ EVs membrane should be disrupted mechanically, thermally or chemically, to release the biomarkers such as RNA of the virus/DNA of human/ biomarkers in extracellular vesicle  Purification and target isolation We usually need to remove the impurities from the biofluid sample containing the target of interest. For example, we need to isolate circulating tumor cells from red blood cells and white blood cells of bloodstream, enabling their analysis. 11 https://www.youtube.com/watch?v=U792BDDNzEs 12 Chapter 2 Biomolecular detection assays  Biomolecular detection assay is an analytic procedure in laboratory medicine, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be in form of any molecular biomarkers discussed previously. Here, we categorize biomolecular detection assays into three main groups based on the employed detection mechanisms.  Biocatalytic assays  Affinity-based assays  Nucleic acid hybridization-based assays  Before elaborating the mechanism of each assay, their applications in the field of disease diagnosis and health monitoring will be discussed 13 Chapter 2 Biomolecular detection assays  The different types of biomolecular assays are widely being used to analyze different types of molecular biomarkers in human body or environment for diagnosis or monitoring purposes.  Biomolecular detection assays are powerful tools to either detect or measure molecular biomarkers indicative of different types of health conditions including:  Infectious diseases  Chronic/organ-related diseases  Cancers 14 Chapter 2 Biomolecular detection assays  Infectious diseases Disorders caused by organisms, such as bacteria, viruses, fungi, or parasites.  The existence of infectious agents, especially viruses and bacteria, can be usually recognized using Biomolecular detection assays through: 1) Detection of structural proteins, locating inside the organism or covering the outer membrane 2) Identification and amplification of a specific part of their genomes (DNA or RNA)  Commonly-used biomolecular detection assays for infectious diseases are: Affinity-based assays and Nucleic acid hybridization-based assays 15 https://www.youtube.com/watch?v=hTWUV6azGXE 16 Chapter 2 Biomolecular detection assays  Chronic/organ-related diseases These type of health conditions can be in the form of autoimmune diseases, heart diseases, kidney and liver dysfunction, ect.  The existence of chronic/organ-related diseases or their risk of progression can be determined through: 1) Measuring the level of protein biomarkers and metabolites in body fluids and compare to their normal levels in healthy controls *In biochemistry, a metabolite is an intermediate or end product of metabolism. The term metabolite is usually used for small molecules.  Commonly-used biomolecular detection assays: Affinity-based assays and Biocatalytic assays  Cancers  The existence or risk of cancer can be determined through: 1) Identification and amplification of a specific gene 2) Either presence or measuring abnormal level of relevant protein biomarkers and microRNA in biofluids.  Commonly-used biomolecular detection assays: Affinity-based assays and Nucleic acid hybridization-based assays 17 Chapter 2 Review  Biomolecular detection assays:  Biocatalytic assays  Affinity-based assays  Nucleic acid hybridization-based assays  Different types of health:  Infectious diseases  Chronic/organ-related diseases  Cancers 18 Chapter 2 Biomolecular detection assays Biocatalytic assays  This method depends on reaction of the target analyte with a recognition element such as enzymes, whole cells, or tissue slices, resulting in production of detectable outcome such as electroactive reporters  In this approach, enzymes are being primarily used due to their high biocatalytic activity and specificity. Sensing devices using enzymes as the recognition element often have relatively simple designs and do not require expensive instrumentation.  The enzyme provides the selectivity for the sensing platform and catalyzes the formation of detectable products for detection. 19 Chapter 2 Biomolecular detection assays Biocatalytic assays: glucose detection via glucose oxidase (GOx) (Chronic/organ-related diseases)  Personal blood glucose monitoring devices are the most successful commercial application of biocatalytic assays enzyme target analye Juska, V. B., & Pemble, M. E. Product of the reaction (2020). Sensors  Considering that the enzyme glucose oxidase (GOx) is specific to glucose, in this method, the oxidation of glucose takes place in the presence of GOx, oxygen (O2) and water (H2O) to form gluconic acid and hydrogen peroxide (H2O2). The hydrogen peroxide is then electrochemically oxidized at the anode of an electrochemical probe, producing an amperometric signal (current) proportional to the concentration of glucose in the sample 20 Chapter 2 Biomolecular detection assays Biocatalytic assays: Cholesterol detection via cholesterol oxidase (ChOx) (Chronic/organ-related diseases)  The Paper-based test strip mainly consists of cerium oxide nanoparticles (nanoceria), as the hydrogen peroxide (H2O2)-dependent color-changing nanozymes, and cholesterol oxidase (ChOx). This system has been developed for convenient colorimetric determination of cholesterol  In the presence of cholesterol, ChOx catalyzes its oxidation to produce H2O2, which forms peroxide complex on the nanoceria surface and induces visual color change of the nanoceria- embedded paper from white/light yellow into intense yellow/orange, which was conveniently quantified with an image acquired by a conventional smartphone with the ImageJ software. Nguyen, P. T., Kim, Y. I., & Kim, M. I. Colorimetric Cholesterol Test Strip (2020). Frontiers in chemistry 21 Chapter 2 Biomolecular detection assays Biocatalytic assays: Uric acid detection via uricase (Chronic/organ-related diseases) Enzymatic colorimetric detection of uric acid  Primarily, in the presence of oxygen, uricase catalyzed specifically uric acid to produce hydrogen peroxide. Next, under the catalysis of molybdenum disulfide (MoS2) nanoflakes, hydrogen peroxide, using hydroxyl radical group (OH ), oxidized the 3,3′,5,5′- tetramethylbenzidine (TMB, a colorless agent) to the oxidized TMB (oxTMB, blue). Wang, X., Yao, Q., Tang, X., Zhong, H., Qiu, P., & Wang, X. (2019). Analytical and bioanalytical chemistry Chapter 2 Biomolecular detection assays Biocatalytic assays: Uric acid (Chronic/organ-related diseases)  The change in color from colorless to blue is related to concentration of UA, which was visible to the naked eyes and measured with UV-vis spectrophotometer. With the increasing of UA concentration (0.5–200 μM), the absorbance of solutions at 652 nm also increased. the color of the solution has also changed visible to the naked eye (colorless → blue). Chapter 2 Biomolecular detection assays Affinity-based assays  Many biochemical analytes of interest are not amenable to detection by enzymatic assays due to the lack of sufficiently selective enzymes being available for the analyte or the analyte not being commonly found in living systems. That is when Affinity-based approaches are considered as an alternative method.  Affinity-based assays employ non-reactive approach, in which specific binding interaction between the analyte and biological recognition element (BRE) is converted into a measurable signal by a transducer  Affinity-based approach exploit selective binding of certain BREs toward specific target species for triggering measurable/detectable outcome. The biomolecular recognition process is governed primarily by the shape and size of the receptor pocket and the ligand of interest (the analyte). Such an associative process is governed by thermodynamic considerations (in contrast to the kinetic control exhibited by biocatalytic systems) 24 Chapter 2 Biomolecular detection assays Affinity-based assays Affinity-based assays can be mainly categorized based on the types of the BRE used in the assay  Antibody-based assays  Aptamer-based assays  Molecularly imprinted polymer (MIP)-based assays 25 Chapter 2 Biomolecular detection assays Antibody  Antibodies have unique patterns of amino acids that can only bind to target antigens with a molecular sequence that provides complementary charges and noncovalent bonds.  Antibodies can be classified into two primary types (monoclonal and polyclonal) by the means in which they are created from lymphocytes 26 Chapter 2 Biomolecular detection assays Antibody  Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is the whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the immune system of human being. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen 27 Chapter 2 Biomolecular detection assays Antibody  Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs). Unlike polyclonal antibodies, which are produced in live animals, mAbs are produced in vitro using tissue-culture techniques. 28 Chapter 2 Biomolecular detection assays Antibody-based assay  The general format of the immunoassay involves coating a solid phase (swab or strip) with the antigen-specific monoclonal antibody and then blocking any unbound sites using a suitable buffer. The coated and blocked swab/strip is incubated with the clinical specimen collected from the patient. The antigen present in the sample would then specifically bind to the monoclonal antibody and other non-specific unbound materials are washed off, after which, the purified BsMAb is used as the detection antibody. As one arm of BsMAb can recognize the antigen, it would bind to the antigen forming a sandwich. Given that BsMAb is already tagged with HRPO, addition of the enzyme substrate TMB results in the formation of blue color that can easily be identified. 1. A Bispecific Antibody Based Assay Shows Potential for Detecting Tuberculosis in Resource Constrained Laboratory Settings 2. 2. Bispecific Antibodies for Diagnostic Applications 29 Chapter 2 Biomolecular detection assays Antibody-based assay: troponin I (Chronic/organ-related diseases)  A sample containing cardiac troponin I (cTnI) mixed with detection antibody- conjugated gold nanoparticles (AuNPs) is added to a capture antibody-coated sensor surface (glass slide) and the formation of the antibody-cTnI- antibody sandwich is detected by digitally counting the binding of the individual gold nanoparticles to the sensor surface in real time using a bright-field optical imaging setup together with a differential imaging algorithm. Wang, Y., Yang, Y., Chen, C., Wang, S., Wang, H., Jing, W., & Tao, N. (2020). ACS sensors, Chapter 2 Biomolecular detection assays Antibody-based assay: E.coli (infectious diseases)  First anti-E. coli antibodies conjugated magnetic beads were used to selectively capture and isolate E. coli from the contaminated water. The isolated bacteria were then co-encapsulated with fluorescently-labeled anti-E. coli antibodies in pico-liter droplets and were analyzed using an automated fluorescence microscope. The detection process required only 8 h of sample collection, pre- concentration, capturing and detection. Golberg, Alexander, et al. PloS one 9.1 (2014) 31 Chapter 2 Biomolecular detection assays Antibody-based assay: E.coli (infectious diseases)  All the steps of the immunoassay are performed inside the flow-through detection cell except for the electrochemical detection step that is performed onto a screen-printed electrode. Bacteria are trapped inside the flow-through detection cell as they cannot pass the filtration membrane. Streptavidin- Horseradish Peroxidase (SA-HRP) binds to the biotinylated anti- E. coli antibodies. In the presence of hydrogen peroxide (H2O2), HRP oxidizes Amplex Red to resorufin. resorufin is redox active and can be detected electrochemically. Chorti, Parthena, et al. Sensors and Actuators B: Chemical 351 (2022) Chapter 2 Biomolecular detection assays Aptamer  Aptamers are short single-stranded oligonucleotides (SSDNA or RNA), which are made of typically 20–80 nucleotides. Aptamers tend to form stable tertiary structures, resulted from combination of secondary structures (short helical arms and single-stranded loops), to specifically bind the target Target biomarkers  Cells  Bacteria  Macromolecules  Small molecules Types of interactions  hydrogen bonding  electrostatic interactions  hydrophobic effect  π–π stacking  van der Waals forces 33 Chapter 2 Biomolecular detection assays Aptamer  Aptamers are produced via a nucleic acid aptamer selection technology, called SELEX (systematic evolution of ligands by exponential enrichment), in which optimized sequences with increased affinity and specificity are isolated from an oligo sequence pool through several rounds of selection Some of the advantages of aptamers  Easy production at large scale  low cost  Stability (pH, temperature)  reproducibility  Can be modified with various functional groups for immobilization purposes 34 Chapter 2 Biomolecular detection assays Aptamer SELEX method mainly consists of the following three steps: selection, partitioning and amplification. Before the selection, a library of oligonucleotides is synthesized, which generally contains up to 1015 different unique sequences. Each unique sequence contains random bases (20–50 nt) flanked by two conserved primer binding sites, which are used for PCR amplification by annealing primers. In the selection step, the library is incubated with target molecules for the indicated time. After incubation, the unbound sequences are separated from those that bound by different methods. The target-bound sequences are amplified by PCR (DNA SELEX) or reverse transcription PCR (RNA SELEX). The PCR products, being a new sub-pool, are utilized for the next round of selection. After several selection rounds, the enriched sequences are sequenced, and their binding abilities are further evaluated. Generally, it takes from weeks to months to obtain specific aptamer candidates, and the hit rates are low. 35 Chapter 2 Biomolecular detection assays Aptamer-based assay  Signal-on aptamer-based cocaine detection. (a) A fluorophore-labeled aptamer is immobilized onto a solid surface. (b) A quencher-labeled short complimentary strand is introduced, decreasing fluorescence. (c) Unlabeled cocaine binds competitively to the aptamer, displacing the quencher and recovering fluorescence. Which is proportion to the concentration of the target 36 Chapter 2 Biomolecular detection assays Aptamer-based assay: TNF-a (Chronic/organ-related diseases)  The biosensor is based on conformational changes of the aptamer. Binding event of the target analyte is presumed to change the distance between redox reporters and electrodes, generating detectable electrochemical signals.  This is a schematic representation of the TNF -a aptasensor with Methylene Blue (MB) as a redox label. In the absence of TNF-a (tumor necrosis factor), aptamer hairpins remain folded with MB reporters in proximity to the electrode, ensuring efficient electron transfer (e) and a measurable faradaic current. Upon target binding, the redox tag moves further away from the electrode and the redox current decreases. Redox reaction of MB Liu, Ying, Qing Zhou, and Alexander Revzin.." Analyst 138.15 (2013): 4321- 37 4326. Chapter 2 Biomolecular detection assays Aptamer-based assay: Cancerous cell (Cancers) aptamer-based fluorescence probe (AAFP)  aptamer TLS11a binds with high affinity to cancer cells. one short DNA sequence (C-strand) was added to each end. The two C-strands were complementary to each other, and the 5' C-strand was conjugated to the fluorophore FAM, while the 3' C-strand was conjugated to the quencher Eclipse. In the absence of a target, the two C-strands hybridized into a hairpin structure, bringing the fluorophore and quencher together and minimizing background fluorescence. Upon target binding, the two C-strands separated, strongly increasing FAM fluorescence signals. Lai, Zongqiang, et al. Oncology reports (2017) Chapter 2 Biomolecular detection assays MIP  Molecular imprinting method is based on the polymerization of a functional monomer and a cross-linker around a template molecule. At first, a pre-complex is formed between a template molecule and a functional monomer and then the polymerization is carried out around the pre- complex with initiator and cross-linker addition. Finally, the template molecule is removed to generate three-dimensional cavities for specific recognition in several times 39 Chapter 2 Biomolecular detection assays MIP-based assay: SARS-CoV- 2 nucleoprotein (infectious diseases) Raziq, Abdul, et al. Biosensors and Bioelectronics (2021)  This is an MIP-based assasy for detection of SARS-CoV-2 nucleoprotein (ncovNP).  The ncovNP sensor was prepared by modification of Au- thin film electrode with ncovNP-MIP film generated from poly-m-phenylenediamine (PmPD) as a functional monomer.  The formation of selective molecular cavities for ncovNP in the polymer (PmPD) can be envisaged via the formation of hydrogen bonds between the sterically accessible protonacceptor groups of the protein (e.g. polar amino acids and sugar residues) and NH2 groups of mPD in the prepolymerization complex.  The chip was connected with a portable readout and the capability of such a sensor system to selectively recognize the target analyte (ncovNP) was studied through differential pulse voltammetry (DPV) in the presence of a redox pair. Chapter 2 Biomolecular detection assays MIP-based assay: H-FABP (Chronic/organ-related diseases)  A hybrid structure of gold nano/micro-islands (NMIs), and MIP was used to to develop an electrochemical biosensor for detection of heart fatty acid binding protein (H-FABP).  Ortho-phenylenediamine (o-PD), the solution containing functional monomers, presents a good biocompatibility behavior against proteins and can be easily electropolymerized on gold electrodes  The MIP layer was Sanati, Alireza, et al. ACS sensors (2021) electropolymerized on NMI modified electrodes and exploited toward detection of HFABP. NMIs feature a high surface area for deposition of MIP Chapter 2 Biomolecular detection assays MIP-based assay: H-FABP (Chronic/organ-related diseases)  The MIP surface seems to be rougher  After washing the electrode with the relative to non imprinted polymer non- optimal solution, the imprinted sites imprinted polymer (NIP), which was are created fabricated without using H-FABP as the template protein. Chapter 2 Biomolecular detection assays Nucleic acid hybridization-based assays  In Simple words, when two single-stranded nucleic acid molecules of complementary base sequence form a double-stranded hybrid the process is known as Nucleic Acid Hybridization  A hybridization assay comprises any form of quantifiable hybridization i.e. the quantitative annealing of two complementary strands of nucleic acids  Nucleic acid sequences are commonly identified by well-established DNA hybridization- based techniques such as polymerase chain reaction (PCR) and Loop-mediated isothermal amplification (LAMP)  While RT-PCR remains the most sensitive diagnostic tool for SARS-CoV-2 detection, LAMP can be considered as a viable alternative to PCR for diagnostic and surveillance. 43 Chapter 2 Biomolecular detection assays LAMP 44 Video 45 Chapter 2 Biomolecular detection assays LAMP  Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction. The LAMP system principally employs four core primers, namely FIP (forward inner primer), BIP (backward inner primer), F3 (forward primer) and B3 (backward primer) to recognize six different regions of the target sequences. A strand-displacing DNA polymerase initiates synthesis and 2 specially designed primers form “loop” structures to facilitate subsequent rounds of amplification through extension on the loops and additional annealing of primers. DNA products are very long (>20 kb) and formed from numerous repeats of the short (80–250 bp) target sequence, connected with single-stranded loop regions in long concatamers. These products are not typically appropriate for downstream manipulation, but target amplification is so extensive that numerous modes of detection are possible. Real-time fluorescence detection using intercalators or probes, lateral flow and agarose gel detection are all directly compatible with LAMP reactions. Instrumentation for LAMP typically requires consistent heating to the desired reaction temperature and, where needed, real-time fluorescence for quantitative measurements. Due to the high rate of product formation within a short period of time (10–15 minutes), LAMP has detection sensitivity comparable to or in many cases, exceeding that of real-time quantitative PCR (qPCR)-the most common “gold standard” technique in most molecular diagnostic assays. Chapter 2 Biomolecular detection assays LAMP  Unlike PCR, loop-mediated isothermal amplification (LAMP) does not require thermal cycling, hence overcoming the logistical challenge of using complex equipment.17 A simple incubator or water bath which is readily available in most endemic communities could be used to perform LAMP. Furthermore, LAMP produces more DNA in a shorter time than PCR, and, most importantly, the enzymes and reagents used in LAMP are more robust and can be stored and transported in less ideal conditions than PCR.  The results of the amplification can be confirmed using different methods, such as changes in turbidity caused by magnesium pyrophosphate precipitate, changes in fluorescence using intercalating dyes, DNA probes with gold nanoparticles, pH indicators, or gel electrophoresis followed by UV detection.  For example, this LAMP kit contains phenol red, a pH indicator that changes color from pink to yellow due to the formation of pyrophosphate ions produced during amplification. Chapter 2 Biomolecular detection assays LAMP: Influenza viruses (infectious diseases)  polymerase enzyme produces protons and subsequently leads to decreased pH in the presence of extensive DNA polymerase activity during LAMP reaction, thus facilitating real-time and simple detection of amplicons as observed by a change from pink to yellow color in the reaction solution  A change in color from pink to light yellow indicated a positive reaction while negative reactions retained a pink color Ahn, Su Jeong, et al. BMC infectious diseases (2019)  Positive RT-LAMP reactions resulted in a color change of phenol red pH indicator from pink to yellow due to decreased pH in the presence of extensive DNA polymerase activity. Thus, results could be directly observed by naked eyes. Chapter 2 Biomolecular detection assays LAMP: E. coli and Salmonella (infectious diseases)  This platform employs electrochemical nucleic acid amplification approach for the detection of E. coli and Salmonella  methylene blue (MB) was used as the DNA-binding redox probe because it is efficiently intercalated into the DNA double helix by van der Waals, hydrogen bonding, and p-p stacking interaction. The MB probe is far more suitable and attractive for use in real- time electrochemical LAMP because of its strong ds-DNA intercalating ability. Huang, Tsung‐Tao, et al. Electroanalysis (2018)  If MB is in a free state, the redox currents are relatively strong. However, when MB intercalates into the DNA minor groove, the MB redox signals are significantly reduced. As the ds-DNA products of LAMP increase, the redox peak currents of MB decrease Chapter 2 Biomolecular detection assays LAMP: E. coli and Salmonella (infectious diseases)  This is an integrated paper/polymer system for on-chip RT-LAMP and colorimetric detection of Prostate cancer antigen3 (PCA3), a tumor marker highly overexpressed in prostate cancer. Wang, Lin-Xiang, et al. Talanta (2020).  A sponge-like polyvinyl alcohol (PVA) pad was used as an amplification unit, on which RT- LAMP reagents and sample RNA were loaded. The RT-LAMP product was injected into the colorimetric detection zone by compressing the sponge-like amplification pad using a stick with a defined protrusion.  Squeezing the reaction solution out for flow into the detection zone, where paper discs containing calcein (5.0mM) and MnCl2(0.1mM) were placed.  The on-chip colorimetric detection of RT-LAMP was inspired by the inhibition of manganese on the fluorescence signal of Calcein. The fluorescence of Calcein can be quenched by manganese ions, while pyrophosphate (P2O74−), the by-product of LAMP assay, can form a complex with manganese ions, displacing the Calcein and resulting in bright fluorescence Chapter 2 Biomolecular detection assays Comparison between nucleic acid hybridization-based assays  Colorimetric and florescent approaches are the most common methods used for detection of LAMP 51 Chapter 2 Biomolecular detection assays DNA-Based BioSensors S.S. Mahshid et.al. J. Am. Chem. Soc. 2015, 137, 50, 15596–15599 52