Journal of Analytical & Bioanalytical Techniques
Open Access

Analytical biochemical techniques; chromatography; ELISA

Introduction

One of the most widely used analytical biochemical techniques is chromatography, which separates molecules based on their chemical and physical properties. High-performance liquid chromatography (HPLC) and gas chromatography (GC) are commonly used [1- 6] to separate and quantify small molecules such as amino acids, nucleotides, and metabolites. HPLC is also used for protein separation and purification. Another important analytical biochemical technique is electrophoresis, which separates molecules based on their size and charge. Gel electrophoresis is commonly used for separating proteins and nucleic acids. Polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) are used to separate proteins, while agarose gel electrophoresis is used for nucleic acid separation. Spectroscopy is another essential analytical biochemical technique that involves the interaction of light with matter. Ultraviolet-visible (UV-Vis) spectroscopy is used to measure the absorbance of molecules such as nucleic acids and proteins, while fluorescence spectroscopy is used to detect and quantify molecules such as fluorophores and proteins. Enzyme-linked immunosorbent assay (ELISA) is a powerful analytical biochemical technique used to detect and quantify proteins or antibodies. ELISA is widely used in diagnostics, drug discovery, and basic research. Mass spectrometry (MS) is a highly sensitive and versatile analytical biochemical technique used to identify and quantify molecules based on their mass-to-charge ratio. MS is used for protein identification, metabolomics, and lipidomics. Analytical biochemical techniques refer to a set of methods used to identify, quantify, and analyze the chemical and physical properties of biological molecules such as proteins, nucleic acids, and metabolites. These techniques are essential for understanding the molecular mechanisms that govern biological processes, as well as for the development of new diagnostic tools, therapies, and drugs. Analytical biochemical techniques are widely used in fields such as biochemistry, molecular biology, pharmacology, and biotechnology. The development of analytical biochemical techniques has been driven by the need to understand the complex and dynamic nature of biological systems at the molecular level. The biochemical molecules that make up these systems are highly diverse and complex; requiring sophisticated analytical methods to study them. The development of these techniques has been facilitated by advances in technology, such as the miniaturization of instruments, the development [1-4] of more sensitive detectors, and the advent of high-throughput methods. In this context, the field of analytical biochemical techniques is constantly evolving, with new techniques and technologies emerging regularly. These innovations have enabled researchers to study biological systems at an unprecedented level of detail, leading to new insights into the molecular mechanisms underlying diseases and the development of more effective diagnostic and therapeutic approaches. In this introduction, we will provide an overview of some of the most commonly used analytical biochemical techniques and their applications. We will also highlight some of the emerging trends and innovations in the field (Table 1).

Table 1: Quantitative data table from ELISA: ELISA can be used to quantitatively measure the amount of a specific protein or other biomolecule in a sample. An example of a data table from an ELISA assay is shown below:

Materials and Methods Analytical Biochemical Techniques

The choice of materials and methods used in analytical biochemical techniques depends on the specific research question and the biochemical molecules being studied. However, there are some common materials and methods [4-9] that are widely used in the field. Materials commonly used in analytical biochemical techniques include buffers, reagents, solvents, and standards. Buffers are used to maintain a constant pH and stabilize biochemical molecules during experiments. Reagents are used to detect, identify, and quantify specific biochemical molecules. Solvents are used to extract, purify, and analyze biochemical molecules. Standards are used to calibrate instruments and validate results. The methods used in analytical biochemical techniques can be broadly classified into separation, detection, and quantification methods. Separation methods are used to separate biochemical molecules based on their chemical and physical properties. Chromatography is a widely used separation method that involves the separation of molecules based on their affinity for a stationary phase (such as a column) and a mobile phase (such as a solvent). High-performance liquid chromatography (HPLC), gas chromatography (GC), and ion-exchange chromatoraphy (IEC) are commonly used in the field. Detection methods are used to detect the presence of biochemical molecules. Spectroscopy is a commonly used detection method that involves the interaction of light with matter. Ultraviolet-visible (UV-Vis) spectroscopy is used to measure the absorbance of molecules such as nucleic acids and proteins, while fluorescence spectroscopy is used to detect and quantify molecules such as fluorophores and proteins. Quantification methods are used to measure the concentration of biochemical molecules. Enzyme-linked immunosorbent assay (ELISA) is a commonly used quantification method that involves the use of antibodies to detect and quantify proteins or other biomolecules. Mass spectrometry (MS) is another commonly used quantification method that measures the mass-to-charge ratio of molecules and is used for protein identification, metabolomics, and lipidomics. In addition to these methods, there are emerging techniques and technologies in the field of analytical biochemical techniques, such as microfluidics, biosensors, and computational methods. These innovations are enabling researchers to study biochemical molecules with greater sensitivity, accuracy, and speed.

In summary, the choice of materials and methods in analytical biochemical techniques depends on the specific research question and the biochemical molecules being studied. Separation, detection, and quantification methods are commonly used in the field, with emerging techniques and technologies offering new possibilities for advancing our understanding of complex biological systems.

Analytical Biochemical Techniques Types of Techniques

There are many different types of analytical biochemical techniques, each with its own strengths and limitations. Some of the most commonly used types of techniques are:

Chromatography: Chromatography is a separation technique used to separate mixtures of compounds based on their chemical and physical properties. High-performance liquid chromatography (HPLC), gas chromatography (GC), and ion-exchange chromatography (IEC) are commonly used in the field.

Electrophoresis: Electrophoresis is a technique used to separate charged molecules, such as proteins and nucleic acids, based on their charge and size. Gel electrophoresis and capillary electrophoresis are commonly used in the field [6-9].

Spectroscopy: Spectroscopy is a technique that involves the interaction of light with matter to provide information about the structure, composition, and dynamics of biochemical molecules. Ultraviolet-visible (UV-Vis) spectroscopy, fluorescence spectroscopy, and infrared (IR) spectroscopy are commonly used in the field.

Mass spectrometry: Mass spectrometry (MS) is a technique used to measure the mass-to-charge ratio of molecules. It is used for protein identification, metabolomics, and lipidomics.

Enzyme-linked immunosorbent assay (ELISA): ELISA is a technique used to detect and quantify proteins or other biomolecules using antibodies.

Polymerase chain reaction (PCR): PCR is a technique used to amplify DNA sequences, allowing for the detection and identification of specific genes and mutations.

Microfluidics: Microfluidics is a technology used to manipulate and analyze small volumes of fluids. It is used for applications such as single-cell analysis and point-of-care diagnostics.

Biosensors: Biosensors are devices that detect and quantify biological molecules. They are used for applications such as medical diagnostics and environmental monitoring.

Computational methods: Computational methods are used to analyze large datasets and model complex biochemical systems. They are used for applications such as drug discovery and personalized medicine. These techniques and others are used in combination to address complex biochemical problems and advance our understanding of biological systems.

Applications on Analytical Biochemical Techniques

Analytical biochemical techniques have a wide range of applications in many fields, including

Biomedical research: Analytical biochemical techniques are used to study the molecular basis of diseases, such as cancer, diabetes, and neurodegenerative disorders. Techniques such as ELISA, PCR, and mass spectrometry are used to detect and quantify specific biomolecules associated with these diseases.

Drug discovery: Analytical biochemical techniques are used to identify and characterize potential drug candidates. High-throughput screening methods, such as mass spectrometry and fluorescence-based assays, are used to screen large libraries of compounds for their ability to interact with specific targets.

Environmental monitoring: Analytical biochemical techniques are used to monitor the levels of pollutants, toxins, and other harmful substances in the environment. Biosensors, chromatography, and mass spectrometry are commonly used in environmental monitoring.

Food science: Analytical biochemical techniques are used to analyze the composition and quality of food products. Techniques such as chromatography, spectroscopy, and electrophoresis are used to identify and quantify specific molecules, such as proteins, carbohydrates, and fats.

Forensic science: Analytical biochemical techniques are used to analyze biological evidence, such as DNA and proteins, in forensic investigations. Techniques such as PCR and mass spectrometry are used for DNA and protein analysis, respectively.

Agriculture: Analytical biochemical techniques are used to improve crop yields and quality. Techniques such as PCR and enzyme assays are used to detect and quantify plant pathogens, while chromatography and spectrophotometry are used to analyze nutrients in soil and plant samples. Overall, analytical biochemical techniques play a critical role in advancing our understanding of biological systems and improving human health and the environment.

Limitations on Analytical Biochemical Techniques

Analytical biochemical techniques have several limitations, including

Sensitivity: Some techniques, such as ELISA and PCR, can be highly sensitive, but they may not be sensitive enough to detect low levels of biomolecules in complex biological samples. This can result in false negative results.

Specificity: Analytical biochemical techniques can be highly specific, but they may not be able to distinguish between closely related molecules. For example, mass spectrometry may not be able to distinguish between two molecules with similar mass-to-charge ratios.

Sample preparation: Analytical biochemical techniques often require extensive sample preparation, which can be time-consuming and costly. Sample preparation can also introduce artifacts or contaminants that can interfere with the accuracy of the results.

Equipment and expertise: Many analytical biochemical techniques require specialized equipment and expertise to perform and analyze the data. This can limit their accessibility to smaller laboratories or institutions with limited resources.

Interference: Analytical biochemical techniques can be subject to interference from other molecules or contaminants in the sample. For example, fluorescence-based assays can be affected by autofluorescence from the sample or background fluorescence from the environment.

Complexity: Biological samples are often complex mixtures of many different molecules, which can make it difficult to isolate and analyze specific biomolecules of interest. This can lead to false positive or false negative results. Overall, it is important to carefully consider the strengths and limitations of analytical biochemical techniques when selecting an appropriate method for a particular application. Researchers should also carefully validate their results and consider multiple lines of evidence to confirm their finding.

Future scope of Analytical Biochemical Techniques

Analytical biochemical techniques continue to evolve, with new technologies and applications emerging in various fields.

Some of the future scopes of analytical biochemical techniques include

Single-cell analysis: Analytical biochemical techniques are increasingly being used for single-cell analysis, allowing researchers to study cellular heterogeneity and diversity in greater detail.

Personalized medicine: Analytical biochemical techniques are being used to develop personalized medicine approaches, based on an individual’s unique genetic and molecular characteristics.

Metabolomics: Analytical biochemical techniques are being used to study the metabolome, the set of small molecules present in a cell, tissue, or organism. Metabolomics can provide insights into disease mechanisms and biomarkers for disease diagnosis.

Nanopore sequencing: Nanopore sequencing is a new technique that uses nanopores to directly sequence DNA or RNA molecules. This technique has the potential to revolutionize genomics and personalized medicine.

Point-of-care diagnostics: Analytical biochemical techniques are being developed for point-of-care diagnostics, enabling rapid and accurate diagnosis of diseases in remote or resource-limited settings.

Bioinformatics: Analytical biochemical techniques generate large amounts of data, which can be analyzed using bioinformatics tools to extract meaningful insights. As bioinformatics continues to advance, it will become increasingly important for analyzing and interpreting analytical biochemical data. Overall, analytical biochemical techniques will continue to play a critical role in advancing our understanding of biological systems and improving human health and the environment. The development of new technologies and applications will allow researchers to tackle new challenges and make new discoveries in the years to come.

Conclusion

Analytical biochemical techniques are essential for understanding the molecular mechanisms underlying biological processes and diseases. Chromatography, electrophoresis, spectroscopy, ELISA, and MS are some of the most commonly used techniques in the field. The continued development and integration of these techniques, along with emerging technologies, will enable researchers to gain a deeper understanding of complex biological systems and develop new therapeutic interventions.

Acknowledgement

The authors are appreciative of the JSPS KAKENHI project’s support (No. 26420267).

Competing Interest

The authors say they have no competing interests.

1. Kim E, Kim J, Choi I, Lee J, Yeo WS, et al. (2020) Organic matrix-free imaging mass spectrometry. BMB reports 53:349.

Indexed at, Google Scholar, Crossref

2. Wang Y, Han Y, Hu W, Fu D, Wang G (2020) Analytical strategies for chemical characterization of bio‐oil. Journal of separation science 43:360-371.

Indexed at, Google Scholar, Crossref

3. Ishii K, Zhou M, Uchiyama S (2018) Native mass spectrometry for understanding dynamic protein complex. Biochim Biophys Acta Gen Subj 1862:275-286.

Indexed at, Google Scholar, Crossref

4. Takeo E, Sasano R, Shimma S, Bamba T, Fukusaki E, et al. (2017) Solid-phase analytical derivatization for gas-chromatography–mass-spectrometry-based metabolomics. Journal of bioscience and bioengineering 124:700-706.

Indexed at, Google Scholar, Crossref

5. Micalizzi G, Vento F, Alibrando F, Donnarumma D, Dugo P, et al. (2021) Cannabis Sativa L.: A comprehensive review on the analytical methodologies for cannabinoids and terpenes characterization. Journal of Chromatography A 1637: 461864.

Indexed at, Google Scholar, Crossref

6. Han Y, Hu W, Fu D, Wang G (2020) Analytical strategies for chemical characterization of bio‐oil. Journal of separation science 43:360-371.

Indexed at, Google Scholar

7. Hubert P, Chiap P, Crommena J, Boulanger B, Chapuzet EN, et al. (2002) The SFSTP guide on the validation of chromatographic methods for drug bioanalysis: from the Washington Conference to the laboratory.Anal Chim Acta 391:135–48.

Indexed at, Google Scholar, Crossref

8. Hartmann C, Smeyers VJ, Massart DL (1999) Validation of bioanalytical chromatographic methods.J Pharm Biomed Anal 17:193–218.

Indexed at, Google Scholar, Crossref

9. Zhoua S, Songb Q, Tangb Y, Weng N(2005) Critical Review of Development, Validation, and Transfer for High Throughput Bioanalytical LC-MS/MS Methods.Curr Pharm Anal 55:3–14.

Google Scholar, Crossref