Diagnostics is a valuable scientific field, contributing daily to the lives of patients. It is through diagnosis that patients gain access to life-saving treatments for a variety of illnesses, from cancer, infectious, neurodegenerative and genetic diseases. It is paramount that the development of successful diagnostic methods is ever-evolving and peptides are the revolutionary tools paving the way in advancing diagnostics and medicine.

Why Peptides?

It is the flexibility of peptides that makes them an ideal tool in both imaging and non-imaging diagnostics. This and the fact that peptides can be:

• naturally degraded and cleared by the human body
• penetrate cells and tissues without inducing an immune response
• target and bind to specific targets within the body
• easily synthesized to display desired properties

Their strong specificity can be exploited for the accurate detection of very small concentrations of target molecules, a key asset in the early diagnosis and monitoring of certain conditions. Examples of diagnostic technologies where peptides are used are Enzyme-Linked Immunosorbent Assays (ELISAs), Radioimmunoassays (RIA), Microarray Technology, Biosensors, Liquid Chromatography-Mass Spectrometry (LC-MS), Positron Emission Tomography (PET), Single-Photon Emission Computed Tomography (SPECT), Multiple Reaction Monitoring (MRM) and Microfluidics.

Our team of global peptide specialists can provide readily available diagnostic peptides in our product catalog or we can even help design, identify and manufacture the most suitable peptide for your diagnostic application using our expert custom synthesis technologies. Visit our diagnostics webpage and read our article to find out more.

Imaging Diagnostics

When evaluating different methods of imaging in diagnostics, it is clear that those that are peptide-based offer several significant advantages over traditional imaging methods. Namely, they exhibit high specificity, can be designed to target a wide range of molecules, and are also less invasive and more cost-effective. It is no surprise that their use in imaging diagnostics is becoming ever more popular. In particular, peptide-based probes have become a highly successful tool in modern imaging diagnostics.

Peptide-Based Probes

Peptides are versatile and can be easily synthesized into a ‘peptide-based probe’ through modifications such as the addition of a label. First, peptides with specificity to your target are selected for. These peptides can be uncovered through techniques such as peptide library screening or phage display.

Radioactive isotopes, such as technetium-99m (Tc-99m), when added to a specific peptide, can be used as radioligands in nuclear medicine imaging. These radioligands can specifically target cancer cells or other cells of interest, by binding to receptors that are typically overexpressed on the surface of these cells during disease states. For example, somatostatin analogues labeled with Tc-99m can be used in scintigraphy to identify and locate tumors, especially in neuroendocrine tumors, which express somatostatin receptors. Other examples of radioactive isotopes of metals currently used in Nuclear Medicine are: In-111, Y-90, Lu-177 and Ga-68. At Biosynth we have an excellent track record in synthesizing precursors for radiolabeled peptides suitable for a wide range of applications, including diagnostics, theranostics and pharmaceuticals. Contact us for more details.

Biosynth’s state-of-the art peptide facilities

Molecular Imaging

Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT) are two of the most common molecular imaging techniques. Both techniques rely on the use of labelled peptides to specifically and accurately show the distribution of disease.

Positron Emission Tomography

PET was shown to be a clinically useful imaging tool in the 1980s and 1990s within the fields of oncology and neurology. It relies on the injection of radioactive tracers into the patient’s body. The tracer is typically a molecule that mimics a specific biological process or substance such as glucose. Once inside the body, the radiotracer emits positrons, which are positively charged particles and the antimatter counterpart of electrons. When a positron encounters an electron they annihilate each other resulting in the release of 511 keV γ-photons (two gamma rays) that travel in opposite directions. This release of gamma rays from the targeted area is then directly detected using a PET scanner, consisting of a ring of detectors that surround the patient. This is termed ‘coincidence detection.’ Advanced computer algorithms process the data collected by the detectors and reconstruct a detailed image of the distribution and concentration of the radiotracer within the body. Analysis of this PET image gives a nuclear medicine specialist information about the metabolic activity or function of the organs and tissues being studied, allowing any abnormalities to be identified.

One such example of a commonly used tracer is the glucose analog fluorine-18 (18F) fluoro-deoxyglucose (FDG), (6-Fluoro-6-deoxyglucose FF69747). Glucose is transported into cells for metabolism, an upregulated process in areas of cancer in the body. As a glucose analog, [18F] FDG is also taken up into cells and because this analog has a radioactive Fluorine instead of the hydroxyl group needed for the steps of metabolism, this analog is prevented from undergoing any further reactions in the cell. This makes [18F] FDG perfect for use in oncology PET scans where the increased cellular uptake of analog [18F] FDG in areas of cancer, show up as a ‘bright spot’, allowing tumors and metastasis to be identified. Other examples of radiotracers used in PET scans are: 68-gallium DOTA peptide which is used in the detection of neuroendocrine tumors and C-labelled metomidate (metomidate, FAA37720) used to detect adrenocortical tumors.

Hospital PET scan

PET scans are not solely limited to oncology, they can be used in neurology and cardiology. In Alzheimer’s disease, [18F] FDG can also be used as a radiotracer in PET scans as there is a decrease in the brain metabolism in Alzheimer’s patients. Furthermore, the characteristic amyloid-beta (Aβ) plaques can be detected using florbetaben F18, Florbetapir F18 and flutemetamol F18. Protein kinase C (PKC) demonstrates its involvement in memory maintenance in Alzheimer disease through promoting amyloid precursor protein cleavage. In the early stages of Alzheimer’s disease, it has been found that levels of PKC are reduced. Given this, an inhibitor of PKC, Enzastaurin (FE22698) can be radiolabeled with carbon-11 and used in PET imaging. Verapamil (FV02354) which is a substrate of P-Glycoprotein, involved in the clearance of Aβ in the brain, can be also radiolabelled with carbon-11 and used in PET scans to show P-Glycoprotein has reduced expression in patients over 75 years.

SPECT

SPECT, like PET, is a nuclear imaging technique; however, a key difference is that SPECT scans use single-photon-emitting radiotracers like technetium-99m or iodine-123. Technetium can be linked to hexamethylpropyleneamine oxime (HMPAO), a blood flow tracer taken up by the brain tissue during metabolism. This ligand is therefore highly successful in showing the health of brain activity and can be used to indicate dementia. When investigating cardiovascular disease and myocardial perfusion SPECT is a highly beneficial diagnostic method. A tracer called sestamibi, made up of technetium and 6 methoxyisobutylisonitrile (MIBI), is used, as MIBI can travel through mycocardial capillary membranes.

Non-imaging Diagnostics

Non-imaging technologies diagnose disease based upon analysis of disease-specific molecules in patient biofluids such as urine, saliva, serum and EDTA plasma.

ELISAs

ELISA assays can identify and quantitatively measure the concentration of a target antigen or antibody in a biological fluid sample. They are reliant upon the specific interaction of an antibody and its antigen and require a method of detection, such as an enzyme-linked conjugate.

ELISAs can be used in both a research and a clinical setting and are particularly beneficial in diagnosing viral diseases such as SARS-CoV-2 (COVID-19), HIV and Yellow Fever. They can also be designed to diagnose autoimmune disorders and cancer. Vasoactive intestinal peptide (FV73664), diphtheria toxin (XD182184) and the COVID-19 spike protein (30-2018), are prime examples of peptide-based diagnostics probes used in ELISAs. Additionally, these assays can monitor the progression of diseases, including metabolic bone diseases like osteoporosis and Paget’s diseases. This is achieved through the quantitative measurement of markers associated with bone turnover, such as Bone Alkaline Phosphatase (BAP).

ELISAs have further illustrated their dominance in the diagnostic field through their ability to contribute to cancer diagnostics. One ELISA has been designed for the detection of mammaglobin in breast cancer patient’s blood (Galvis-Jiménez, 2013). Furthermore, in the diagnosis of diabetes an antibody sandwich ELISA has been developed for the detection of C-peptide (PCP-3725-PI), which is increased in diabetic patients (Lv, 2019).

Even though peptide-based ELISAs are highly advantageous in that they have high specificity and sensitivity and can target a wide range of molecules, the stability of the peptide needs to be controlled. Additionally, factors such as cross-reactivity and interferences from other molecules in the sample or assay conditions need to be considered and investigated. As a result, ELISAs are carefully validated and standardized before use in diagnostic applications. See our ELISA technical methods bulletin for a guide to ELISAs and their parameters.

Microarray technology

Another non-imaging diagnostic method orchestrating the use of peptides is microarrays. Synthetic peptide probes, which may be unique peptide biomarkers for a disease of interest, are adsorbed onto nitrocellulose-coated glass slides. These are then exposed to specimens, possibly in the form of a serum sample or a cellular extract, providing favorable conditions for antigen-specific antibodies to be captured. One of the main applications and advantages of using peptide microarray technology is that a large pool of different peptide biomarkers which are specific to a particular disease can be analyzed. Peptide microarrays are especially useful in:

Antibody profiling: to detect the presence of specific antibodies in a serum sample, such as in autoimmune disease or infectious diseases.

Tissue profiling: to detect the presence of specific proteins in a tissue sample, such as in cancer diagnosis.

Peptide microarray illustration

At Biosynth, we can identify unique peptide disease biomarkers with the greatest specificity for an antibody or molecule of interest using our specialist peptide library technology. We even have the capabilities to manufacture your optimum diagnostic peptide to a large scale or improve its application properties through modifications such as the addition of labels or our proprietary CLIPS™ technology.

Visit our custom peptide libraries page to find out more about further applications such as epitope mapping, drug discovery and protein characterization.

Biosensors

Leland C. Clark Jr, now known as the ‘father of biosensors,’ produced the first biosensor used to detect oxygen in 1956. Since then, biosensors have been developed for use in the clinical diagnosis and prognosis of diseases. They can even be used in drug discovery. A biosensor is made up of the following components:

• A target molecule that needs to be detected – this could be an antibody produced by a patient.

• A molecule, termed a bio receptor, that is specific and will be able to recognize the target molecule – peptides can be used as a bio receptor.

• A transducer has the ability to convert energy from the peptide molecule recognizing the target molecule into an electrical or optical signal.

• An electric circuit to amplify and covert signals into a digital form.

• A method to display the results so that they can easily be interpreted, for example a liquid crystal display.

• Generally, a signal measured is proportional to the concentration of the target molecule binding to the peptide bio receptor.

Biosensors are commonly used in point-of-care (POC) diagnostics, which are diagnostic tests that can be performed at or near the site of patient care, such as in a doctor’s office, clinic, or at home. POC diagnostics are particularly useful in resource-limited settings, where access to laboratory facilities is limited. There are different types of biosensors used in diagnostics:

Electrochemical biosensors: These biosensors use an electrode to detect changes in the electrical current caused by the presence of a target molecule. They can be used to detect a wide range of molecules, such as the presence of HIV-anti-p24 antibodies, glucose, lactate, and creatinine (FC01718).

Optical biosensors: These biosensors use light to detect changes in the properties of a sample caused by the presence of a target molecule. They can be used to detect a wide range of molecules, such as enzymes, antibodies, and nucleic acids. In the diagnosis of HIV, bifunctional colorimetric or fluorescence assay short peptides originating from HIV-1 p17 proteins are tagged with a sensitive dye.

Piezoelectric biosensors: These biosensors use the mechanical properties of a sample to detect changes caused by the presence of a target molecule. They can be used to detect a wide range of molecules, such as proteins, nucleic acids, and small molecules. One example is where amino acids 567-613 of the gp41 protein (30-039001) are polymerized onto a quartz crystal microbalance and these are used to detect anti-HIV antibodies.

Liquid Chromatography-Mass Spectrometry

Liquid chromatography-mass spectrometry (LC-MS) is a powerful and highly sensitive analytical technique at the forefront of diagnostic technologies. It is critical in discovering and measuring levels of biomarkers prevalent in disease states. LC-MS methods can also be multiplexed, meaning that only a single assay is needed to quantify a protein instead of several assays. Peptides can be used in LC-MS to diagnose disease by detecting and quantifying specific peptide biomarkers in biological samples. Mass-shifted, stable-isotope-labeled peptide calibrators are required for this method.

First, a biological sample is taken from a patient and processed through separation processes like centrifugation and liquid chromatography, in order to isolate the target peptide of interest. During chromatographic separation the sample is introduced into a column containing a stationary phase which is specific to the peptide you intend to capture. Those peptides and molecules which are not complementary to the stationary phase travel through the column. The separated peptides are then introduced into the mass spectrometer, where they are ionized and their mass-to-charge ratio (m/z) is measured. The mass spectrometer can detect and identify the different peptides in the sample, based on their m/z values, and can also provide information about their relative abundance.

Stable-isotope-labelled peptide calibrators are spiked into samples, and the ratio of labelled and unlabeled peak heights determines the quantification. Currently this method, known as the stable isotope dilution method (SID) is the gold-standard in multiple reaction monitoring (MRM) and selective reaction monitoring (SRM).

Mass Spectrometry at Biosynth

As leaders in isotope labelled peptides since 2001 and the pioneers of the first complete collection of peptide and antibody SIS products and services, Neptune™, our team at Biosynth can give you expert advice and support in designing your peptide for use in diagnostic proteomics.

Diagnostic Peptides at Biosynth

At Biosynth, we are proud to manufacture a diverse range of custom diagnostic peptides with exceptional quality, precision, and versatility. Whether you need peptides for immunoassays, biosensors, microarrays, or other diagnostic applications, we have the expertise to synthesize peptides of varying lengths, purities, and modifications. Our team of experts will work closely with you to design and produce diagnostic peptides that precisely match your project’s objectives, providing you with the tools to drive your research forward. Visit our diagnostic peptides page to discover how our peptides can empower your diagnostic projects, enabling you to drive advancements in healthcare and improve patient outcomes. Also see our biologics webpage and catalog products, host to raw materials for in vitro diagnostics.

Contact Biosynth today to discuss your diagnostic peptide requirements