Analytical Methods in Biochemistry

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the primary method used to measure the scattering of light in turbidimetry?

A spectrophotometer is used to measure the scattering of light in turbidimetry.

How does nephelometry differ from turbidimetry in terms of sensitivity and measurement?

Nephelometry is more sensitive than turbidimetry and measures scattered light at specific angles.

What is a commonly used light-absorbing particle in lateral flow assays?

Gold particles are commonly used as light-absorbing particles in lateral flow assays.

What is a characteristic feature of organic fluorophores that makes them suitable for multiplex assays?

Each organic fluorophore has a specific emission wavelength. Signup and view all the answers

How are fluorescent proteins like GFP utilized in microscopy?

They are primarily used as fusion proteins. Signup and view all the answers

What technology can be used to measure reflectance in particles for point-of-care testing?

Reflectance spectroscopy is used to measure the signal produced in point-of-care tests. Signup and view all the answers

What is the main function of the enzyme in a fluorometric ELISA?

The enzyme converts a non-fluorescent substrate into a fluorescent product. Signup and view all the answers

What role does the enzyme play in the enzyme-linked immunosorbent assay (ELISA)?

The enzyme in ELISA converts a non-detectable substrate into a detectable product. Signup and view all the answers

Why is reflectance spectroscopy considered difficult to standardize?

Reflectance spectroscopy is difficult to standardize due to variations in angle of incidence and surface area. Signup and view all the answers

Name one drawback of using fluorescent labels in assays.

Self-quenching occurs at high concentrations. Signup and view all the answers

Which lanthanide is most commonly used in immunoassays?

Europium (Eu) is most commonly used. Signup and view all the answers

What are the limitations of lateral flow immunoassays (LFIA) in terms of sensitivity?

Lateral flow immunoassays are not very sensitive compared to other quantitative tests. Signup and view all the answers

What is the impact of particle size on the observations made in nephelometry?

Different analyte sizes produce different angles of scattered light in nephelometry. Signup and view all the answers

What role does an organic ligand play in the use of lanthanide chelates?

The ligand delivers energy to the ion and shields it from water. Signup and view all the answers

What is a notable advantage of using lanthanide labels in assays?

They have long-lived fluorescence. Signup and view all the answers

What is the effect of the plastic backing in assays using fluorescent labels?

It contributes to background signal noise. Signup and view all the answers

What kind of light source is used in the fluorescence measurement method?

A pulsed light source, specifically a xenon lamp, is used for excitation. Signup and view all the answers

What is the role of nanoparticles filled with europium in fluorescent assays?

They enhance signal intensity and shield europium chelates from water to avoid the quenching phenomenon. Signup and view all the answers

How does the concentration of a sample affect the dynamic range in measurements?

High concentrations of a sample reduce the dynamic range of the measurement. Signup and view all the answers

What are the advantages of using time-resolved fluorescence measurement?

It provides long-lasting fluorescence and helps distinguish between signal and background noise. Signup and view all the answers

What is one method of obtaining a noncompetitive immunoassay using europium nanoparticles?

Coating particles with streptavidin allows them to react with a biotinylated antibody. Signup and view all the answers

What is the size of the europium nanoparticles used for labeling in assays?

The nanoparticles have a size of approximately 107 nm. Signup and view all the answers

What is the significance of having a longer excitation wavelength compared to the emission wavelength in assays?

This configuration allows for higher sensitivity and simplifies the detection device. Signup and view all the answers

How do UCNPs improve assay capabilities when using whole blood?

UCNPs allow for assays to be conducted without the need for separation from plasma or serum. Signup and view all the answers

What advantage do dopant ions provide in multiplexing assays with UCNPs?

Dopant ions allow for different emission wavelengths while maintaining the same excitation wavelength. Signup and view all the answers

Identify one difficulty associated with the coating of biomolecules on particle-based reporters.

Coating is challenging because particles must be made hydrophilic and chemically suitable for biomolecule coupling. Signup and view all the answers

What is photobleaching and which type of fluorophores are most susceptible to it?

Photobleaching is the loss of fluorescent properties due to photon exposure, with organic fluorophores being the most affected. Signup and view all the answers

Define chemiluminescence and its advantages in assay technology.

Chemiluminescence is light emission from chemical reactions, offering higher sensitivity and lower cost due to no need for excitation light. Signup and view all the answers

What is a key disadvantage of chemiluminescence assays?

The main disadvantage is that it involves a one-time reaction, limiting repeated measurements. Signup and view all the answers

What is required for the effective measurement of short-lived chemiluminescent signals?

A sample cuvette must be positioned close to the detector immediately after the reaction starts. Signup and view all the answers

What is the basic principle behind IMMUNO-PCR?

IMMUNO-PCR utilizes a sandwich-type immunoassay with oligonucleotides labeled antibodies, followed by amplification and quantification of the analyte concentration through qPCR. Signup and view all the answers

How does the Direct ELISA (SiMoA) detect analytes?

Direct ELISA detects analytes by using beads linked to antibodies with beta-galactosidase that convert a substrate into a fluorescent product, which is then measured. Signup and view all the answers

What is the role of the average number of enzymes per bead (AEB) in the Direct ELISA?

AEB accounts for the likelihood of multiple analytes binding to a single bead, impacting the accuracy of analyte concentration measurements. Signup and view all the answers

Why is Poisson distribution important in quantifying analyte concentrations?

Poisson distribution is important as it allows for the calculation of the likelihood of events occurring, helping to estimate the number of enzymes based on an average known events. Signup and view all the answers

What is the significance of reporter recycling in IMMUNO-PCR?

Reporter recycling enhances signal amplification and increases the sensitivity of the assay without requiring additional enzymes. Signup and view all the answers

How does the Direct ELISA minimize the risk of signal leakage during measurement?

The assay minimizes signal leakage by sealing the wells containing beads with oil or another substance to prevent mixing of the liquid phases. Signup and view all the answers

What factors can lead to inaccurate measurements in the Direct ELISA?

Inaccurate measurements can occur if the average number of enzymes per bead (AEB) is too high, leading to most beads being 'on', which complicates quantification. Signup and view all the answers

Describe the relationship between analyte concentration and fluorescence in Direct ELISA.

As analyte concentration increases, the number of beads producing fluorescence also increases, allowing for quantification based on the proportion of 'on beads'. Signup and view all the answers

Study Notes

### Turbidimetry and Nephelometry

Turbidimetry measures the amount of light transmitted through a solution.
Nephelometry measures the scattered light at specific angles.
Turbidimetry is easier to use but less sensitive.
Nephelometry is more sensitive but more complex and requires expensive equipment.

Lateral Flow Assays

Lateral flow assays (LFIA) often use gold particles as labels.
The size of the gold particle determines the wavelength of light it absorbs.
Lateral flow assays are not very sensitive but can be made semi-quantitative with specific devices.
Reflectance spectroscopy can be used to quantify the signal produced in LFIA.

ELISA

ELISA stands for enzyme-linked immunosorbent assay.
The enzyme attached to the tracer antibody is the label.
The enzyme converts a non-detectable substrate into a detectable product, which can be colored, fluorescent, or chemiluminescent.

Fluorescent Labels

Organic fluorophores contain aromatic groups and conjugated bonds.
Each fluorophore has a specific emission wavelength, making them useful for multiplex assays.
Fluorescent proteins, such as GFP and proteins from red algae, can also be used as labels in microscopy techniques.

Fluorometric ELISA

The enzyme in fluorometric ELISA converts a non-fluorescent substrate into a fluorescent product.
Beta galactosidase, horseradish peroxidase (HRP), and alkaline phosphatase are common enzymes used in fluorometric ELISA.
Fluorescent labels have a narrow stroke shift and a short half-life, requiring instantaneous measurement.
Self-quenching can occur at high label concentrations.

Lanthanide Chelates

Lanthanide chelates are rare earth elements like europium (Eu), terbium (Tb), samarium (Sm), and dysprosium (Dy).
They are used in immunoassays because they have intense fluorescence, a wide stoke shift, sharp emission bands, and long-lived fluorescence.
Lanthanides are often attached to a molecule like an antibody using an organic ligand.
There are two types of chelating structures: fluorescent and non-fluorescent chelates.

Non-fluorescent Chelates

Have a short-lived background signal followed by a counting window.
Are easy to separate the excitation light from the emission light.
Require complex instrumentation for measurement.
The instrumentation includes a pulsed light source, spectral system, optical system, sample compartment, spectral selection system, detector, and read out system.

Particle-based Fluorescent Reporters

Types of particle-based fluorescent reporters:
- Europium nanoparticles
- Quantum dots
- Upconverting nanoparticles (UCNPs)
Europium nanoparticles are filled with europium chelates and shielded from water to avoid quenching.
Quantum dots are semiconductor nanocrystals.
UCNPs can be used with whole blood and are suitable for multiplexing assays.
Particle-based reporters are difficult to coat with biomolecules, can aggregate, and can limit antibody options due to their size.

Photobleaching

Photobleaching is the destruction of the fluorescent property of a fluorophore by the same photons it absorbs and emits.
Some fluorophores are more resistant to photobleaching than others.
Organic fluorophores are the most susceptible, while TRF chelates and particle-based reporters are more resistant.

Chemiluminescence

Chemiluminescence is the emission of light produced by chemical reactions.
Chemiluminescence assays are highly sensitive due to the lack of initial light absorption.
Chemiluminescence is a one-time reaction, requiring immediate measurement.
The instrumentation is simple and requires a functional electrode.

Immuno-PCR

Immuno-PCR is a qPCR reaction with a sandwich immunoassay using labeled oligonucleotides.
The analyte concentration is directly related to the number of amplified sequences.

Direct ELISA

Direct ELISA is also called SiMoA, Single Molecule Array, or Quanterix assay.
The principle is that ELISA is performed on a bead linked with a beta-galactosidase enzyme.
The beads are made to fall into an array, with theoretically only one bead per well.
The fluorescence emanated by the wells is measured, and the number of "on beads" is counted.
The analyte concentration is determined using the Poisson distribution.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz explores various analytical techniques used in biochemistry, including turbidimetry, nephelometry, lateral flow assays, and ELISA. Understand the principles behind each method and their applications in laboratory settings. Test your knowledge of fluorescence and its role in labeling techniques.