10.2: Diagnostic Biotechnology

Biotech Focus

Conventional laboratory techniques for diagnosing metastatic breast cancers include a combination of imaging, tissue analysis, and biomarker testing. Tumor biopsies are tested for common breast cancer biomarkers like the ER marker (estrogen receptor) and the HER2 marker (human epidermal growth factor receptor 2; also called HER2/neu) using immunological methods, like FISH, and the BRCA-2 marker using molecular techniques such as DNA sequencing. These methods can be time-consuming and expensive to perform.

However, researchers at Hacettepe University and Abant İzzet Baysal University in Turkey have created a potential means of rapidly diagnosing HER2-positive breast cancer using a unique biosensor called a quartz crystal microbalance (QCM). A QCM is a piezoelectric sensor, means that it uses a thin quartz crystal that oscillates at a specific frequency. When molecules accumulates on the crystal, the change in mass of the crystal decreases its oscillation frequency. This frequency shift is directly proportional to the mass of the material that has bound to the crystal surface, even at the nanogram or even picogram level. To detect breast cancer, researchers can coat the crystal with nanoparticles bound to transferrin, a iron-binding protein. As a result of their enhanced metabolic activity, aggressively-growing breast cancer cells are known to overexpress the receptor to transferrin (i.e., the transferrin receptor). As a result, breast cancer cells will bind to this sensor via their transferrin receptor and change its oscillation frequency.

To test the QCM biosensor, breast cancer cells were added to a transferrin-coated crystal to allow for binding. The oscillation frequency was measured as an indication of the concentration of this cancer biomarker. Researchers found that the QCM biosensor function correlated well with the number of transferrin receptors on the cells, making this a potential diagnostic tool for identifying the early stages of breast cancer, monitoring its progression, or evaluating a patient's response to therapy.

To learn about recent advances in using a QCM in detecting breast cancer, include coating the crystal's nanoparticles with antibodies to the HER2/neu biomarker, check out this article.

Introduction

Diagnostic biotechnology involves the use of biological tools and techniques to detect and/or measure biomarkers associated with diseases, genetic conditions, or infections. The exploitation of these tools and techniques has led to the industrial production of biological products for diagnostic purposes. These products are now used in clinical laboratories around the world. A biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often used to indicate a normal or abnormal process, identify a disease, or evaluate a response to treatment. In medicine, biomarkers are usually molecules found in blood, other body fluids, or in tissues.

Biomarkers are classified according to their use and include:

• Diagnostic biomarkers: used to diagnose a condition like a disease
  • e.g., estrogen and progesterone receptors used in the diagnosis of breast cancer
• Prognostic biomarkers: markers that assess the progression of a diagnosed disease, regardless of treatment
  • e.g., Brain Natriuretic Peptide (BNP) expression used to assess heart failure prognosis
• Predictive biomarkers: markers that predict a response to a specific treatment
  • e.g., HER2 receptor used to predict response to chemotherapies
• Monitoring biomarkers: markers that track disease progression or treatment response over time
  • e.g., troponin synthesis used to monitor heart disease
  • e.g., creatinine levels used to monitor kidney disease
• Susceptibility biomarkers: markers that identify an increased risk of developing a disease
  • e.g., BRCA1/BRCA2 mutations used to assess the risk of breast cancer
  • e.g., LDL levels used to assess atherosclerosis
• Pharmacodynamic/Response biomarkers: markers that indicate a biological response to a drug in order to assess whether the drug is working
  • e.g., decrease in hemoglobin A1c (HBA1c) levels in response to diabetes treatment
• Safety biomarkers: markers that indicate toxicity of adverse effects of a specific treatment
  • e.g., liver enzymes monitored for drug-induced liver injury

Here are some key areas used in diagnostic biotechnology:

1. Molecular Diagnostics
2. Immunodiagnostics
3. Point of Care (POC) Diagnostics

Learning Objectives

Diagnostic biotechnology uses biological tools and techniques to diagnose diseases, disorders, and conditions. At the end of this page, you will be able to:

• List and define the key areas of diagnostic biotechnology
• Explain next-generation sequencing
• Explain the DNA microarray
• Explain Fluorescent In Situ Hybridization (FISH)
• Describe the structure of an antibody
• Explain the different types of ELISAs
• Explain how the "sandwich" ELISA is performed and how this protocol differs from the direct and indirect ELISA
• Define point-of-case diagnostics (POC)
• Explain how a lateral flow assay (LFA) works
• Define biosensor and explain how one works in POC
• Explain how the glucose monitor works as a biosensor
• Explain the lab-on-a-chip

Molecular Diagnostics

Molecular diagnostics is a field of medical testing that uses molecular biology techniques to detect and measure specific DNA, RNA, or protein markers in cells or tissues. These tests are used to diagnose diseases, predict risk, guide treatment, and monitor health conditions at the molecular level.

Key techniques used in molecular diagnostics are:

1. Polymerase Chain Reaction (PCR): amplifies specific DNA or RNA sequences
   • used to detect the presence of specific genetic markers associated with diseases and infections
   • to read more about PCR, go to Chapter 3.2: Replication of DNA
2. Real-Time PCR (qPCR): quantifies the amount of genetic material in a sample
   • used to quantitate the expression level of genetic markers
   • to read more about PCR, go to Chapter 3.2: Replication of DNA
3. Next-Generation Sequencing (NGS): sequences entire genomes or targeted regions for mutations
4. Microarrays: analyzes the expression levels of thousands of genes at once
   • two major types: DNA microarrays and protein microarrays
   • to read about protein microarrays, go to Chapter 5.6: Proteomics
5. In situ hybridization (e.g., FISH): detects specific DNA or RNA in cells

Next Generation Sequencing

While modern day Sanger sequencing instruments are still used by the majority of research facilities to quickly sequence a small DNA construct, they are limited by the relatively small number of sequencing reactions that can be analyzed at one time (e.g. 8 to 96). Next-generation sequencing (NGS) is a powerful platform that enables the sequencing of thousands to millions of DNA molecules simultaneously, thus allowing for the sequencing of an entire genome in one day. In NGS, large-scale DNA libraries are first created by shearing the genome into smaller fragments, denaturing them into single strands and attaching specialized oligonucleotide sequences, called "adaptors" to each end of the DNA fragment (Figure \(\PageIndex{1}\)). These adaptors allow for hybridization of the DNA library fragments to complementary sequences called DNA "linkers". These linkers are found coupled to a specialized glass surface, called a flow cell. The adaptors also contain small stretches of sequence that can serve as an "index" to identify the bound fragment once sequencing is complete.

Once bound to their linker, each DNA library fragment is amplified through a multi-step process called bridge amplification. This unique amplification method produces thousands of DNA copies identical to the original bound DNA fragment. Because these copies are located at a specific area of the flow cell, they are called "clusters". The DNA in each cluster becomes the starting point for a subsequent sequencing reaction called Sequencing by Synthesis or (SBS). SBS uses using fluorescently-labeled "terminator" nucleotides that are similar to those used in Sanger sequencing. However, these nucleotides are said to be "reversible". During SBS, a DNA polymerase synthesizes a DNA chain that is complementary to the bound library fragment using these fluorescent nucleotides. The addition of each nucleotide is known as a "round" or "cycle". A camera takes an image after each round in order to detect the fluorescent signal. Following incorporation of the nucleotide, the fluorescent tag and terminator are removed, thus permitting a new round. The process of nucleotide incorporation and terminator/fluorescent tag removal is repeated for 50 to 300 cycles, depending on the fragment size.

At the end of these SBS phase, the fluorescent images from each cluster are converted into a sequence of bases called a "read". The reads from each cluster are analyzed using bioinformatic programs that align the DNA sequences using a reference genome or assemble them de novo into a genomic sequence.

Figure \(\PageIndex{1}\): The Next Generation Sequencing (NGS) workflow. NGS can sequence multiple genomes in a day. (1) DNA Library Production: The genome is sheared into smaller fragments. Each fragment is flanked by two types of adaptors to create a DNA library for sequencing. (2) Bridge Amplification: Through their adaptors, DNA library fragments are bound to complementary DNA oligos located at specific locations on a "flow cell". The bound genomic fragments are then amplified through “bridge amplification” where the DNA strand folds over and binds to an adjacent oligo. A complementary strand is made. Repeated copying produces “clusters” of double-stranded DNA. The DNA is denatured and all reverse strands are removed, leaving the forward strands bound to the cell as “forward strand clusters”. (3) Sequence Detection: The forward strands in each cluster are copied using fluorescently-labeled “terminator” nucleotides. As each nucleotide is incorporated into the copy, a camera detects the fluorescent signal. The fluorescent signal and terminator tag are then removed from the nucleotide, permitting another round of nucleotide incorporation. The process is repeated. (4) Sequence Alignment: The fluorescent signals from each cluster are converted into a DNA sequence called a “read". Sequences from the clusters are aligned by bioinformatic programs to produce the complete genome sequence. (Next Generation Sequencing (NGS) Workflow by Patricia Zuk, CC BY 4.0)

NGS offers researchers a high-throughput option by giving labs the capability to sequence the genomes from multiple individuals at the same time, allowing a genomic comparison between normal individuals and those with a disease or disorder. Through this work, diseases and disorders can be linked to specific DNA mutations.

Concept in Action:

Video: Next Generation Sequencing

Microarrays

DNA Microarrays can be used to measure the expression of thousands of genes simultaneously and are often used to compare gene expression between different cell types or conditions. The microarray is a solid surface onto which short DNA sequences, called DNA probes (or probes), are affixed in a grid pattern. Each DNA probe corresponds to a specific gene in an organism's genome. To perform a microarray, total RNA is extracted from a sample being studied and converted into complementary DNA (cDNA). A fluorescent label is then added to the cDNA. The labeled cDNA sample is added to the microarray, where the cDNA molecules will bind to their complementary probe sequence. The unbound cDNA is washed away, leaving only the labeled cDNA molecules bound to ther DNA probes. The microarray is then scanned with a laser, exciting the fluorescent labels and capturing the fluorescent signal from each spot. Binding of a cDNA molecule to its probe indicates expression of a specific gene. The intensity of fluorescence at each probe spot indicates the expression level of that gene.

Multiple samples can be analyzed using microarrays, ranging from hundreds of samples analyzed by "high-throughput" platforms (e.g., Affymetrix or Illumina) to just two samples (i.e., a dual-color microarray). In a dual-color microarray, the cDNA from each sample is labeled with a different fluorescent label, such as green for the control sample and red for the experimental sample. The samples are applied to the microarray as described above. The fluorescent color and intensity is measured at each DNA probe spot and then used to compare gene expression levels between the two samples (Figure \(\PageIndex{2}\)).

Figure \(\PageIndex{2}\): The steps of dual-color microarray analysis. (1) RNA Isolation: RNA is isolated from two different samples (RNA Sample #1 and RNA Sample #2). (2) cDNA Synthesis & Labeling: cDNA samples are synthesized from these RNA samples and labeled with different fluorescent molecules (cDNA sample #1 with red probe, cDNA sample #2 with green probe). (3) Application to Microarray: the labeled cDNA samples are added to a microarray slide made of thousands of short DNA sequences (DNA probes). Binding of a labeled cDNA to the complementary DNA probe represents expression of a specific gene. (4) Probe Excitation: Excitation of the fluorescent probes produces fluorescent signals that are detected by a fluorescent reader. Spots with red fluorescence represent gene expression in sample #1. Spots with green fluorescence represent expression in sample #2. Spots with yellow fluorescence (i.e. green plus red) represent gene expression by both samples. Empty spots (gray color) indicate no cDNA binding to a probe and no expression of that gene. (5) Data analysis: quantitation of gene expression levels can be determined by measuring fluorescent levels on the slide. Comparison of expression levels between cDNA samples can also be performed. (Microarray Analysis by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. 2025)

Microarrays allow researchers to assess gene expression profiles across thousands of genes simultaneously. Like NGS, data obtained from DNA microarray analysis can be used to compare the genetic "signatures" of a disease or infection to its healthy counterpart. However, proper data analysis is important since the amount of data generated by a microarray can be significant.

Concept in Action

DNA Microarray Methodology

Fluorescent In Situ Hybridization (FISH)

Fluorescent In Situ Hybridization (FISH) is a molecular technique that uses a fluorescently-labeled probe to detect specific DNA or RNA sequences in a cell or tissue. Because this hybridization occurs within a cell or tissue, it is considered to be "in situ" (i.e., in place). Using a fluorescent microscope, FISH allows scientists to confirm the presence and location of a genetic sequence within the sample. The probes used for FISH can be either DNA or RNA. These DNA or RNA probes are chemically synthesized and are typically designed to be small in length (~20 to 50 nucleotides). The DNA probe is designed to be complementary to a specific DNA sequence in the sample, while an RNA probe is complementary to a specific mRNA or miRNA sequence. Once synthesized, the DNA or RNA probe is then labeled with a fluorescent dye. However, DNA probes can also be labeled with other compounds like fluorescently-bound antibodies. The actual process of FISH will depend on whether the probe is RNA or DNA and how the probes are labeled. To perform FISH, the sample for analysis is prepared by "fixing" the cell or tissue onto a microscope slide (Figure \(\PageIndex{3}\)). Chemicals such as paraformaldehyde or formaldehyde are used. Following fixation, the sample is permeabilized with a detergent to allow for penetration of the probe into the sample and the DNA on the slide is denatured. The probe is allowed to bind, or hybridize, to its target sequence in the sample. Excess probes are washed away and the sample is viewed under a fluorescent microscope.

Figure \(\PageIndex{3}\): Fluorescent in situ hybridization (FISH). (1) Prepare sample on slide: samples a fixed to a microscope slide, permeabilized with detergent and the DNA in the sample denatured. (2) Prepare probe: DNA (or RNA) probes are synthesized to be complementary to a target DNA sequence in the sample and then labeled with a fluorescent probe (green circle). (3) Hybridize labeled probe to denature sample DNA: the probe is hybridized to the sample to allow for the binding of the probe sequence to its complementary target on the sample's chromosome. (4) Visualize fluorescence: fluorescence can be viewed using a fluorescent microscope. The physical location of probe binding to chromosomes (shown in blue) is seen as orange fluorescent dots. (FISH by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2025); image credit: Karyotype of Zebrafish by Freeman et al., CC BY-2.0)

FISH is a fast and specific molecular technique that has numerous medical applications, including the detection of chromosomal abnormalities during pre-natal testing (e.g., Down's syndrome), the diagnosis of cancer (e.g. HER2 in breast cancer), the identification of microbes within a sample, the expression of a gene within a tissue, or the mapping of genes to a specific chromosome. Examples of diseases and syndromes that can be detected using FISH include, acute lymphoblastic leukemia, chronic myelogenous leukemia, Down's syndrome, Cri-du-Chat, and Angelman syndrome. In medicine, FISH is also used to evaluate disease prognosis and remission of a disease like cancer. Treatments can then be tailored to fit the patient.

Immunodiagnostics (Immunoassays)

Immunodiagnostics is a group of diagnostic techniques that use the specificity of antibodies to detect and measure substances in biological samples. An antibody, also known as an immunoglobulin, is a protein produced by the B-cells of the immune system in response to the presence of a foreign substance known as an antigen. Antigens are typically sugars or proteins found on the surface of cells (i.e., bacterial, fungal, mammalian) and viruses. The antibody is made of four polypeptide chains, two heavy chains and two light chains, that link together to form a Y-shape (Figure \(\PageIndex{4}\)). The tips of these four chains is known as the variable region (or antigen-binding site). This region changes its shape from antibody to antibody to "match" a specific antigen, giving the antibody the critical property of specificity. The remaining regions of the antibody, called the constant region, can bind to other immune cells or immune components to heighten the immune response. Once bound to the antigen, the antibody can neutralize the antigen, identify the invading cells for destruction by the immune system, and activate the complement system, enhancing the immune response.

Figure \(\PageIndex{4}\): The structure of an antibody. The antibody is comprised of four polypeptides known as two heavy chains and two light chains. The ends of the light and heavy chains are known as the antigen-binding site or the variable region (shown in yellow). This variable region changes its shape to reflect the specific antigen that binds to this region. (Antibody Structure by Patricia Zuk, CC BY 4.0; modified from Antibody by Fvasconcellos, public domain)

Immunodiagnostic tests are used to identify infectious diseases (e.g., HIV, hepatitis, COVID-19), autoimmune diseases (e.g., rheumatoid arthritis, lupus), and cancer markers (e.g., PSA, CA-125). They are also used in allergy testing and can quantitate substances like hormones (e.g., TSH, insulin) and growth factors (e.g., EGF, VEGF). They are highly specific and sensitive due to the precise interaction between antibodies and antigens. As such, immunodiagnostic tests can detect minute amounts of substances like hormones, proteins, microbes, or antibodies.

Common immunodiagnostic techniques include:

1. ELISA (Enzyme-Linked Immunosorbent Assay) – for detecting antigens or antibodies.
2. Western Blotting – for detecting specific proteins in a sample.
3. Immunofluorescence – uses fluorescent-labeled antibodies to detect targets in cells or tissues.
4. Lateral Flow Assays – like pregnancy tests or rapid COVID-19 tests.
5. Radioimmunoassay (RIA) – uses radioactive isotopes to detect small amounts of substances.

The Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA stands for Enzyme-Linked Immunosorbent Assay. The ELISA is a common laboratory technique that is used to detect and measure an antigen, such as a protein, found in a sample. Samples that are assayed using an ELISA can range from blood and urine (assayed in clinical labs) or cell lysates (analyzed in clinical and research labs). At the end of the ELISA, a "signal" is produced that can be detected and measured. Antigens that can be detected through ELISAs include antibodies, hormones, growth factors, and toxins. ELISAs are used to diagnose diseases (e.g. HIV, hepatitis), detect allergens in food, monitor hormone levels in blood and urine, test for toxins or pathogens in water or food, and measure antibody levels (i.e., titers) in blood following vaccination.

Concept in Action

The ELISA

There are several types of ELISAs (Figure \(\PageIndex{5}\)), including the direct ELISA and indirect ELISA. In both of these ELISAs, the sample is added to a solid surface in order to bind the antigen to it. The direct ELISA uses an enzyme-bound primary antibody to directly bind the antigen. The direct ELISA is quick and easy to perform but produces a low signal intensity as there is only a single type of antibody used. The antigen in the indirect ELISA is bound by a primary antibody, followed by an enzyme-coupled secondary antibody. Because multiple secondary antibodies can bind a single primary antibody, the indirect ELISA produces an amplified signal. It is also a more flexible ELISA because different types of secondary antibodies can be used. The direct and indirect ELISA use an antigen-coated solid support. In contrast, the "sandwich" ELISA starts with a solid support coated with an antibody. In the "sandwich" ELISA, the sample protein to be detected is captured or "sandwiched" in between the antibody-coated surface and the detection antibodies used for quantitation. The sandwich ELISA has high sensitivity and amplification, but non-specific antibody binding can interfere with the results.

Figure \(\PageIndex{5}\): ELISA types. Some of the major types of ELISAs are direct, indirect, and "sandwich". In the direct ELISA, a primary antibody, coupled to an enzyme binds the antigen bound to the solid support. In the indirect ELISA, a primary antibody binds the antigen, which is then bound by the enzyme-coupled secondary antibody. In the "sandwich" ELISA, the solid surface is coated with a "capture" antibody. This capture antibody binds the antigen. The bound antigen is then detected with a primary antibody and enzyme-coupled secondary antibody. For each ELISA type, the antibody-bound enzyme processes a substrate into a signal that can be detected and quantified. (ELISA Types by Patricia Zuk, CC BY 4.0; modified from ELISA Types by Allan.richard5093, CC BY-SA 4.0)

To perform an "sandwich" ELISA, the following steps are taken:

1. Coating: A solid surface, usually a 96-well plate, is coated with an antibody
2. Blocking: Non-specific binding sites on the plate are blocked to prevent background signal
3. Sample addition: The sample is added and binding of the target to the antibody occurs
4. Probing: A target-specific primary antibody is added to the plate to bind and identify the bound target
5. Washing: Excess primary antibody is removed
6. Detection: A secondary antibody that is linked to an enzyme is added to bind the primary antibody
7. Washing: Excess secondary antibody is removed
8. Substrate Addition: A substrate specific for the enzyme is added; the enzyme reacts with the substrate to produce a color change or a fluorescent or luminescent signal
9. Measurement: The intensity of the signal is measured using a spectrophotometer or luminometer; this measurement is proportional to the amount of the target molecule

The procotols for direct and indirect ELISAs are very similar to the "sandwich" ELISA. Each protocol is summarized below in Table \(\PageIndex{1}\).

Table \(\PageIndex{1}\): ELISA Protocols

ELISA Step	Direct ELISA	Indirect ELISA	"Sandwich" ELISA
1. Coating	Coat with sample	Coat with sample	Coat with "capture" antibody
2. Blocking	Yes	Yes	Yes
3. Sample addition	No	No	Yes
4. Probing	Yes, primary antibody coupled to enzyme	Yes; uncoupled primary antibody	Yes; uncoupled primary antibody
5. Washing	Yes	Yes	Yes
6. Detection	No	Yes, secondary antibody coupled to enzyme	Yes, secondary antibody coupled to enzyme
7. Washing	No	Yes	Yes
8. Substrate Addition	Yes	Yes	Yes
9. Measurement	Yes	Yes	Yes

Lateral flow assays

A lateral flow assay (LFA) is a simple, fast, and portable test used to detect the presence of a substance in a liquid sample like water, blood, or saliva. These substances can include proteins, pathogens, or chemicals. The best example of an LFA is a pregnancy test. However, other LFAs include those that will detect viral infections (e.g., the COVID-19 test), bacterial contamination, and the presence of heavy metals in water.

The LFA (Figure \(\PageIndex{6}\)) is made of the following parts:

• a sample pad where the sample is introduced
• a conjugate pad that contains detector molecules, such as antibodies or DNA probes, that mix with the sample
  • the detector molecules are tagged with a visible label
• a nitrocellulose membrane as the core of the LFA
  • multiple membranes could be present with each strip performing a specific function
  • the membrane has a test line that indicates whether the target is present with a physical line and a control line that confirms whether the test worked
• an absorbant pad (wicking pad) that pulls the liquid through the strip and soaks up excess fluid

Figure \(\PageIndex{6}\): The lateral flow assay. The lateral flow assay (LFA) test strip is made of a sample pad, a conjugate pad containing antigen-specific antibodies conjugated to a tag like a gold nanoparticle, a nitrocellulose membrane with an antibody-bound test line, and a wicking pad. The sample (i.e. the analyte) is applied to the sample pad and capillary action draws the sample through the remaining parts of the LFA test strip. The antigens found in the sample bind to the conjugated antibodies in the conjugate pad. If the antigen also binds these antibodies found in the test strip, the conjugated tags will form a physical line and indicate a positive result. (Lateral Flow Assay, no author, public domain)

LFAs are easy to use. The sample is added to the sample pad and the liquid flows along the conjugate pad and into the nitrocellulose pad due to capillary action. If the target substance is present it will bind to the tagged detector molecules. The resulting complexes will stick to the test line found in the nitrocellulose membrane and create a visible line to indicate a positive test result.

Point of Care Diagnostics

The LFA describe above is an example of Point of Care Diagnostics (PCD). PCD are medical tests done at or near the place where the patient is located. PCD allows for rapid diagnostics, rather than waiting for a distant lab to provide results. PCD are relatively easy to perform and can be performed by medical personnel and even the patient themselves. The rapid turn around time gives medical personnel the chance to make quick decisions for the health and well-being of the patient.

Examples of PCD include:

• Lateral Flow Assays (LFAs)
• Glucose Monitors
• Lab-on-a-Chip

Each of these PCD examples provide fast results (often within minutes), are portable and easy to use, require minimal training to operate, and can be used at home, in clinics, ambulances, and even at remote locations where clinics are unavailable.

Lab-on-a-Chip

More advanced PCD are being made possible thanks to lab-on-a-chip (LOC) technology. An LOC is a device that integrates one or several laboratory functions on a single integrated circuit, known as the "chip" (Figure \(\PageIndex{7}\)). This chip is only a few millimeters to a few square centimeters and is designed to achieve automation and high-throughput screening (HTS). LOCs can handle extremely small fluid volumes of blood, urine, or saliva, even down to less than a picoliter (i.e., a trillionth of a liter). The LOC is very similar to an LFA, but can provide more complex analysis.

To use an LOC, a tiny drop of fluid is placed into the chip. Microfluidic channels move the liquid within the chip using either capillary action, electric fields, pressure, or vacuum. As the liquid moves through the chip, it passes through reaction chambers where it mixes with reagents that allow for the detection of targets like DNA, proteins, or bacteria. The results can appear as a visual color change, a fluorescent signal, or an electrical signal that is sent to an computer or a phone app.

Figure \(\PageIndex{7}\): a Microfluidic chip. The microfluidic chip is a key element of lab-on-a-chip devices that are outside hospitals. The chip consists of multiple microfluidic channels and a detection system of metal electrodes on a glass substrate. (Microfluidic Chip by FMNLab, CC BY-SA 4.0)

Like immunodiagnostic tests, LOC technology can diagnose diseases, measure hormones and growth factors, and detect toxins. However, their more advanced technology also allows for the analysis of DNA or RNA (i.e. portable DNA sequencers), blood and urine. LOC technology may soon become an important part of efforts to improve global health, as a part of PCD. In countries with few healthcare resources, LOCs may be able to provide rapid and accurate diagnosis of things like infectious diseases.

Biosensors

A biosensor is a device that uses a biological component, like an enzyme, antibody, or DNA sequence, to detect a specific substance. The biosensor then converts that detection into a readable signal, such as light, color, or an electric signal. The results are usually displayed as a digital reading, a color change or graph.

The main parts of the biosensor (Figure \(\PageIndex{8}\)) are the:

1. Bioreceptor - the biological part that recognizes and binds the target being detected
2. Transducer - converts the biological reaction into a signal
3. Display - shows the result

Figure \(\PageIndex{8}\): The biosensor. The biosensor is made of three components: the bioreceptor for detection of the target, the transducer which converts the detection into a measurable signal, and the display which displays the results (The Biosensor by Patricia Zuk, CC BY 4.0)

Perhaps the most well-known biosensor is the glucose monitor. The bioreceptor in this sensor is an enzyme called glucose oxidase. Glucose oxidase binds glucose and, through oxidation, converts it to gluconolactone and hydrogen peroxide. This enzyme is physically bound to an electrode. The oxidation reaction releases electrons which flow through the electrode as an electrical current. The amount of current flowing through the electrode is proportional to enzyme activity and glucose concentration in the blood.- The result in then displayed as milligrams per deciliter (mg/dL) or millimoles per liter (mmol/L). In addition to the glucose monitor, biosensors are also used in biotechnology research, drug development, environmental monitoring, and food safety.

Key Concepts

Diagnostic biotechnology involves the use of biological tools and techniques to detect diseases, genetic conditions, or infections. Some important concepts to remember

• molecular diagnostics uses molecular tools like PCR and sequencing to associate DNA sequences with diseases, disorders, and infections
• next-generation sequencing can sequence thousands of DNA fragments at a time and sequence a entire genome in a few hours
• DNA microarrays can analyze sequences from multiple samples at the same time
• NGS and DNA microarrays allow researchers to compare normal genomic sequences to those associated with a specific disease, disorder, or infection
• FISH is used to physically identify the location of a specific DNA or RNA sequence in a genome and can be used to diagnose chromosomal abnormalities, cancer, and microbial contamination
• immunodiagnostics like the ELISA or lateral flow assay (LFA) uses antibodies for diagnosis
• the ELISA is used to measure the amount of a specific antigen or antibody in a sample
• the lateral flow assay (LFA) is a test strip embedded with an antigen-specific antibody
• the LFA is used to quickly identify the presence of an antigen in a sample
• point-of-care diagnostics (PCD) are medical tests performed at local site other than hospital, clinic, or research lab
• PCD include the LFA, biosensors, and the lab-on-a-chip
• a biosensor, like a glucose monitor, uses biological component, like an enzyme, antibody, or DNA sequence, to detect a specific substance in a sample
• advanced biosensors can be combined into a lab-on-a-chip capable of performing multiple chemical steps in a diagnosis

Glossary

 

Adaptors - short, synthetic DNA sequences attached to a DNA fragment; used in Next Generation Sequencing

Alignment - the process of arranging DNA or amino acid sequences using regions of sequence overlap

Antibody - a Y-shaped protein produced by B cells of the immune system that recognizes and binds to a specific antigen; also called an immunoglobulin

Antigen - a substance (usually a protein or sugar) that triggers an immune response in the body; often used in vaccines

Bioreceptor - a biological molecule or structure that interacts with a target substance

Capillary action - the movement of liquid through a narrow space, like a thin tube or porous material, due to cohesion between water molecules or adhesion between water and the surface

Capillary gel - a thin, glass tube filled with a polymeric gel; used to quickly separate molecules, like DNA fragments, according to their size and electrical charge

Complementary DNA (cDNA) - DNA that is synthesized from an RNA template (usually mRNA) using the enzyme reverse transcriptase

Computational assembly - the process of aligning DNA fragments into a continuous sequence (i.e., a contig) using computer alignment programs

Dideoxynucleotide (ddNTP) - a DNA nucleotide used in Sanger sequencing that lacks the 3'-OH (hydroxyl) group necessary for continued 5'-to-3' DNA synthesis; used to terminate the elongation of the complementary DNA strand

Fluorescently-labeled - a compound that has a fluorescent dye attached to it

High-throughput screening (HTS) - a method for scientific discovery that uses robotics, data processing/control software, liquid handling devices, and sensitive detectors, to allow a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests; used in drug discovery and the fields of biology, materials science and chemistry

Microfluidics - the science and technology of manipulating small volumes of fluids in channels with dimensions of tens to hundreds of micrometers; typically involves volumes of microliters to picoliters

Microfluidic channels - tiny fluid-conducting pathways etched or molded into microfluidic devices; guide fluid flow precisely for chemical reactions, separation, or detection on diagnostics chips or cards

Next Generation Sequencing (NGS) - a high throughput sequencing method that can sequence several genomes in a day; also known as massive parallel sequencing

Oligonucleotide - a short, single-stranded fragment of a nucleic acid, like DNA or RNA, typically containing between 2 and 50 nucleotides; also called an "oligo"

Point of Care Diagnostics (PCD) - medical tests done at or near the place where the patient is located; also called Point of Care Testing (POCT)

Probe - a single strand of DNA or RNA that is complementary to a nucleotide sequence of interest

Reagent - any chemical or biological substance used in a laboratory test or reaction to cause a chemical reaction or detect a target; includes enzymes, antibodies, dyes, and buffers

Sanger sequencing - a DNA sequencing method that uses dideoxynucleotides (ddNTPs) to terminate the synthesis of the complementary DNA chain; also known as chain termination sequencing

Total RNA - the complete set of RNA molecules extracted from a cell or tissue at a given time; includes mRNA, tRNA, and rRNA

Transducer - a cellular component that converts a biological reaction into a signal