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Thrombin is a multifunctional serine protease that plays an important role in coagulation and anticoagulation processes. Aptamers have been widely applied in biosensors due to their high specificity, low cost and good biocompatibility. This review summarizes recent advances in thrombin quantification using aptamer-based biosensors. The primary focus is optical sensors and electrochemical sensors, along with their applications in thrombin analysis and disease diagnosis.

Circadian desynchrony with the external light-dark cycle influences the rhythmic secretion of melatonin which is among the first signs of circadian rhythm sleep disorders. An accurate dim light melatonin onset (established indicator of circadian rhythm sleep disorders) measurement requires lengthy assays, and antibody affinities alterations, especially in patients with circadian rhythm disorders whose melatonin salivary levels vary significantly, making antibodies detection mostly inadequate. In contrast, aptamers with their numerous advantages (e.g., target selectivity, structural flexibility in tuning binding affinities, small size, etc.) can become preferable biorecognition molecules for salivary melatonin detection with high sensitivity and specificity. This study thoroughly characterizes the structural property and binding mechanism of a single-stranded DNA aptamer full sequence (MLT-C-1) and its truncated versions (MLT-A-2, MLT-A-4) to decipher its optimal characteristics for saliva melatonin detection. We use circular dichroism spectroscopy to determine aptamers' conformational changes under different ionic strengths and showed that aptamers display a hairpin loop structure where few base pairs in the stem play a significant role in melatonin binding and formation of aptamer stabilized structure. Through microscale thermophoresis, aptamers demonstrated a high binding affinity in saliva samples (MLT-C-1F K d  = 12.5 ± 1.7 nM; MLT-A-4F K d  = 11.2 ± 1.6 nM; MLT-A-2F K d  = 2.4 ± 2.8 nM; limit-of-detection achieved in pM, highest sensitivity attained for MLT-A-2F aptamer with the lowest detection limit of 1.35 pM). Our data suggest that aptamers are promising as biorecognition molecules and provide the baseline parameters for the development of an aptamer-based point-of-care diagnostic system for melatonin detection and accurate profiling of its fluctuations in saliva., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Our knowledge of cell- and tissue-specific quantification of phytohormones is heavily reliant on laborious mass spectrometry techniques. Genetically encoded biosensors have allowed spatial and some temporal quantification of phytohormones intracellularly, but there is still limited information on their intercellular distributions. Here, we review nucleic acid aptamers as an emerging biosensing platform for the detection and quantification of analytes with high affinity and specificity. Options for DNA aptamer technology are explained through selection, sequencing analysis and techniques for evaluating affinity and specificity, and we focus on previously developed DNA aptamers against various plant analytes. We suggest how these tools might be applied in planta for quantification of molecules of interest both intracellularly and intercellularly., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tumor-derived exosomes are vital for clinical dynamic and accurate tumor diagnosis, thus developing sensitive and multiple exosomes detection technology has attracted remarkable attention of scientists. Here, a novel herringbone microfluidic device with aptamer-functionalized barcodes integration for specific capture and multiple detection of tumor-derived exosomes is presented. The barcodes with core-shell constructions are obtained by partially replicating the periodically ordered hexagonal close-packaged colloidal crystal beads. As their inverse opal hydrogel shell possesses rich interconnected pores, the barcodes could provide abundant surface area for functionalization of DNA aptamers to realize specific recognition of target exosomes. Besides, the encoded structure colors of the barcodes can be maintained stably during the detection events as their hardish cores are with sufficient mechanical strength. It is demonstrated that by embedding these barcodes in herringbone groove microfluidic device with designed patterns, the specific capture efficiency and synergetic detection of multiple tumor-derived exosomes in peripheral blood can be significantly improved due to enhanced resistance of turbulent flow. These features make the aptamer-functionalized barcodes and herringbone microfluidics integrated platform promising for exosomes extraction and dynamic tumor diagnosis., (© 2022 Wiley-VCH GmbH.)

Aflatoxin M 1 (AFM 1 ) is a common toxin in dairy products that causes acute and chronic human health disorders. Thus, the development of a rapid and accurate AFM 1 detection method is of vital importance for food safety monitoring. This work was to develop a novel electrochemical aptasensor for sensitive and specific determination of AFM 1. The dendritic-like nanostructure was formed on the gold electrode surface by layer-by-layer assembly of gold-silver core-shell nanoparticles modified with DNA conjugates. In the presence of AFM 1 , the specific recognition between AFM 1 and Apt caused the disassociation of the DNA controlled dual Au@Ag conjugates from the surface of the electrode, causing less methylene blue to bind to the surface and weakening the electrochemical signal. The more AFM 1 there is, the weaker the electrochemical signal. Transmission electron microscope results showed that the successfully synthesized Au@Ag nanoparticles exhibited a core-shell structure with Au as core and Ag as shell, and their average diameter was about 30 nm. Under optimal conditions, the electrochemical aptasensor showed a wide detection ranging from 0.05 ng mL -1 to 200 ng mL -1 , and a low detection limit of 0.02 ng mL -1 . Moreover, the proposed strategy has been successfully applied to the detection of AFM 1 in cow, goat, and sheep milk samples with satisfactory recoveries ranging from 91.10% to 104.05%. This work can provide a novel rapid detection method for AFM 1 , and also provide a new sensing platform for the detection of other toxins., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Herein, we present a facile and sensitive fluorescence resonance energy transfer (FRET) aptasensor for the detection of pathogenic bacteria, where antibiotic-functionalized cerium oxide nanoparticles were served as an energy donor and aptamer-modified gold nanoparticles (aptamer-AuNPs) were employed as an energy acceptor. To illustrate the feasibility of this strategy, Escherichia coli ( E. coli ) was examined. The strategy for the detection of E. coli bacteria as a target molecule is described using the FRET pair of azithromycin-functionalized CeO 2 nanoparticles (Azm-CeO 2 NPs) and aptamer-AuNPs. The spectral overlap between these two nanoparticles and Azm and the aptamer binding on the surface of E. coli specifically provides the condition, which leads to the occurrence of the FRET phenomenon. In this way, a good linear correlation between the fluorescence intensity of Azm-CeO 2 NPs and E. coli concentration was obtained in the range of 10-1.5 × 10 5 cfu mL -1 . The detection limit of the proposed method at a signal to noise ratio of 3 (3σ) was estimated to be 1.04 cfu mL -1 . Further, the proposed method was applied to detect E. coli in real samples within 30 min, which indicates the applicability of the proposed method. This method could be used for other pathogenic bacterium recognition or synchronous detection by employing molecules that are particular to the desired bacteria.

Probing pro-metastatic biomarkers is of significant importance to evaluate the risk of tumor metastasis, but spatially selective imaging of such targets in extracellular microenvironment is particularly challenging. By introducing the bilinguality of PNA/peptide hybrid that can speak both peptide substrate and nucleobase-pairing languages to combine with aptamer technology, we designed a smart DNA nanodevice programmed to respond sequentially to dual pro-metastatic targets, MMP2/9 and ATP, in extracellular tumor microenvironment (TME). The DNA nanodevice is established based on the combination of an ATP-responsive aptamer sensor and a MMP2/9-hydrolyzable PNA/peptide copolymer with a cell membrane-anchoring aptamer module. Taking 4T1 xenograft as a highly aggressive tumor model, the robustness of the DNA nanodevice in spatioselective imaging of MMP2/9 and ATP in TME is demonstrated. We envision that this design will enable the simultaneous visualization of multiple pro-metastatic biomarkers, which allows to gain insights into their pathological roles in tumor metastasis., (© 2021 Wiley-VCH GmbH.)

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K D ) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa , we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG 4 -SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

Biosensor technology is considered to be a great alternative in analytical techniques over the conventional methods. Among many recently developed techniques and devices, aptasensors are interesting because of their high specificity, selectivity and sensitivity. Combining aptamer as a biological recognition element with gold nanoparticles (AuNPs) as probe, are becoming more general owing to their beneficial properties, including low cost and ability to analyze specific targets on-site and using naked eye. Hydrogen bonds, nucleic acid hybridization, aptamer-target and antigen-antibody binding, Raman signature, enzyme inhibition, and enzyme-mimicking activity are main different sensing strategies exploited in AuNPs-based optical aptasensors. In this review article, we discussed the recent advances in optical aptasensors with a special emphasis on the catalytic activity property of AuNPs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genetically encoded molecular tools are crucial for live cell RNA imaging, and few are available for endogenous RNA imaging. We develop a new genetically encoded sensor using conformation switching RNA induced fluorogenic proteins that enable multicolor and signal-amplified imaging of endogenous RNAs. The sensor system is designed with an RNA sensing module and a degron-fused fluorescent protein reporter. Target RNA induces conformation switching of the RNA sensing module to form RNA aptamers that stabilize the degron-fused protein for fluorogenic imaging. This sensor is demonstrated for high-contrast imaging of survivin mRNA abundance and dynamics in live cells. Moreover, the sensor system is extended to a multicolor palette by screening fluorogenic proteins of distinct colors, and engineered into a signal amplifier using the split fluorescent protein design. The sensor is further exploited for imaging lncRNA MALAT-1 and its translocation dynamics during mitosis. Our sensor system can afford a valuable platform for RNA imaging in biomedical research and clinical theranostics.

As a new molecular recognition element, oligonucleotide aptamer not only has higher affinity and specificity to target molecules, but also has the advantages of wide recognition range, in vitro synthesis and chemical stability compared with conventional antibodies. Since a kind of screening method termed systematic evolution of ligands by exponential enrichment (SELEX) was reported, scientists have extensively researched the methodology of how to highly and efficiently screen out aptamers from a library consisting of a large number of random oligonucleotides. Certainly capillary electrophoresis-based screening methodologies, including nonequilibrium capillary electrophoresis of equilibrium mixtures, equilibrium capillary electrophoresis of equilibrium mixtures, non-SELEX, ideal-filter capillary electrophoresis, capillary transient isotachophoresis, etc., are revolutionary. Compared with conventional SELEX, these capillary electrophoresis-based methodologies show incomparable advantages such as the single-round screening of aptamers and increased successful screening rate. Methodology studies on the screening process of aptamers are comprehensively reviewed., (© 2021 John Wiley & Sons, Ltd.)

Aptamers are small nucleic acids that fold in a three-dimensional conformation allowing them to bind specifically to a target. This target can be an organic molecule, free or carried in cells or tissues, or inorganic components, such as metal ions. Analogous to monoclonal antibodies, aptamers however have certain advantages over the latter: e.g., high specificity for their target, no to low immunogenicity and easy in vitro selection. Since their discovery more than 30 years ago, aptamers have led to various applications, although mainly restricted to basic research. This work reviews the applications of aptamers in veterinary science to date. First, we present aptamers, how they are selected and their properties, then we give examples of applications in food and environmental safety, as well as in diagnosis and medical treatment in the field of veterinary medicine. Because examples of applications in veterinary medicine are scarce, we explore the potential avenues for future applications based on discoveries made in human medicine. Aptamers may offer new possibilities for veterinarians to diagnose certain diseases-particularly infectious diseases-more rapidly or "at the patient's bedside". All the examples highlight the growing interest in aptamers and the premises of a potential market. Aptamers may benefit animals as well as their owners, breeders and even public health in a "One Health" approach.

Aptamers are short single-stranded oligonucleotides (either DNA or RNA) that can fold into well-defined three-dimensional (3D) spatial structures which enable them to capture their specific target by complementary shape interactions. Aptamers are selected from large random libraries through the SELEX process and only a small fraction of the sequence is involved in direct docking with the target. In this paper, we describe the possible truncation variants of zearalenone (ZEA) aptamer which might be an effective binding region for the target. The originally selected zearalenone (ZEA) aptamer was 80-mer in length and shown to bind the target with a high affinity (K d  = 41 ± 5 nM). Herein, computational docking simulation was performed with 15 truncated variants to determine the predicted binding energy and responsible binding site of the aptamer-analyte complex. The results revealed that 5 truncated variants had binding energy lower than - 7.0 kcal/mol. Circular dichroism analysis was performed on the shortlisted aptamer and the conformational change of aptamers was observed with the presence of an analyte. Aptamer Z3IN (29-mer) was chosen as the most enhanced affinity for its target with a dissociation constant of 11.77 ± 1.44 nM. The aptamer was further applied in the electrochemical aptasensor of ZEA based on an indirect competitive format. The results demonstrated that the truncated aptamer leads to an enhancement of the sensitivity of the biosensor.

The interaction of human epidermal growth factor receptor 3 (HER3) and heregulin (HRG) is involved in resistance to human epidermal growth factor receptor 2 (HER2)-targeted cancer treatment, such as therapies using anti-HER2 monoclonal antibody. Therefore, inhibition of the HER3/HRG interaction is potentially valuable therapeutic target for cancer treatment. In this study, we used in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX) against the extracellular domain of human HER3, and discovered a novel RNA aptamer. Pull-down and bio-layer interferometry assays showed that RNA aptamer discovered specifically bound to HER3 with a dissociation constant (K D ) of 700 nM. Pull-down assays using chemiluminescence detection also revealed that the HER3-binding RNA aptamer inhibited interactions between HER3 and human HRG. These results indicated that the novel HER3-binding RNA aptamer has potential to be used as basic tool in a range of applications involving HER3/HRG interactions, including research, therapeutic, and diagnostic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Biochemical properties and flexibility of nitrogenous bases allow DNA to fold into higher-order structures. Among different DNA secondary structure, G-quadruplexes (tetrapelexes-G4) - which are formed in guanine rich sequences - have gained more attention because of their biological significance, therapeutic intervention, and application in molecular device and biosensor. G4-quadruplex studies categorize into three main fields, in vivo, in vitro, and in silico. The in vitro field includes G4 synthetic oligonucleotides. This review focuses on the G-quadruplex synthetic aptamers structure features and considers the applicability of G4-aptamers for cancer biomarkers detection. Various biosensing methods will be reviewed based on G-quadruplex aptamers for cancer detection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A donor-cell-assisted membrane biotinylation strategy was used to modify small extracellular vesicles (sEVs) while minimizing protein damage, and allowed the sEVs to be loaded onto carriers. Biotinylated programmed death-ligand 1 (PD-L1) positive sEVs were used to select for aptamers from a DNA library. PD-L1 negative sEVs from a homologous cell line were found to remove non-specific aptamer sequences to increase the specificity. After just four rounds, high-affinity aptamers for PD-L1 positive sEVs were selected as novel affinity reagents.

This study was to find the aptamers with high affinity and specificity binding to acute promyelocytic leukemia (APL) NB4 cell line. These aptamers targeted NB4 cells were selected from a random single-stranded DNA (ssDNA) library of systematic evolution of ligands by exponential enrichment (CELL-SELEX). The binding rate of FITC-ssDNA library and NB4 cells was monitored using flow cytometry and fluorescence microscope. After cloned and sequenced, the structure, specificity, and affinity of these candidate aptamers were further analyzed. After a total of 19 rounds of selection, the ssDNA library was enriched and the BR (19.9%) of the 16th round was 12 times of the first round (1.6%). Three enriched aptamers were obtained from 21 positive clones of the 16th round, and the predicted secondary structures of these aptamers were mainly stem-loop. The aptamer CX9 had the highest affinity, and the equilibrium dissociation constant (Kd) was 16.2 nM. The fluorescence intensity of CX9 binding to NB4 cells was stronger than HL60 and K562 cells under fluorescence microscopy. The study indicates that aptamer CX9 exhibits high affinity and specificity with NB4 cells and lay a foundation for the rapid diagnostic method to detect APL with fluorescence-labeled aptamer., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Nucleic acid aptamers for biosensing are developed from a complex ssDNA library through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. Monitoring of SELEX process is crucial for generating high-affinity aptamers. Extant methods for monitoring aptamer selection are either arduous or give false-positive signals, which adversely impact the outcome of selection. We describe a colorimetric, simple and cost-effective, novel method to monitor the progress of in vitro selections. The power of rolling circle amplification (RCA) and inherent Horse Radish Peroxidase (HRP)-mimicking activity of G-quadruplex/hemin DNAzyme were employed to produce a colorimetric signal. A unique extension of DNA population at 3'-OH end by PCR generated concatenated repeats by rolling circle amplification (RCA) reaction. Oxidation of substrate ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) in presence of H 2 O 2 and hemin cofactor produced colorimetric signal. Analysis of the signal generated by the DNA pool bound to their target provided a quantitative measurement of SELEX. We demonstrate the reproducibility and accuracy of the method by evaluating the progress of two discrete selections., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.

As a significant constituent in biosphere, bacteria have a great influence on human activity. The detection of pathogen bacteria is closely related to the human health. However, the traditional methods for detection of pathogenic bacteria are time-consuming and difficult for quantification, although they are practical and reliable. Therefore, novel strategies for rapid, sensitive, and cost-effective detection are in great demand. Aptamer is a kind of oligonucleotide that selected by repeated screening in vitro or systematic evolution of ligands by exponential enrichment (SELEX) technology. Over the past years, owing to high affinity and specificity of aptamers, a variety of aptamer-based biosensors have been designed and applied for pathogen detection. In this review, we have discussed the recent advances on the applications of aptamer-based biosensors in detection of pathogenic bacteria. In addition, we also point out some problems in current methods and look forward to the further development of aptamer-based biosensors for pathogen detection.

Abnormal glycan structures are valuable biomarkers for disease states; the development of glycan-specific binders is thereby significantly important. However, the structural homology and weak immunogenicity of glycans pose major hurdles in the evolution of antibodies, while the poor availability of complex glycans also has extremely hindered the selection of anti-glycan aptamers. Herein, we present a new approach to efficiently screen aptamers toward specific glycans with a complex structure, using a glycosylated peptide as a scaffold. In this method, using peptide-imprinted magnetic nanoparticles (MNPs) as a versatile platform, a glycopeptide tryptically digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection. Alternating positive and negative selection steps were carried out to guide the directed evolution of glycan-binding aptamers. As proof of the principle, the biantennary digalactosylated disialylated N -glycan A2G2S2, against which there have been no antibodies and lectins so far, was employed as the target. With the glycoprotein transferrin as a source of target glycan, two satisfied anti-A2G2S2 aptamers were selected within seven rounds. Since A2G2S2 is upregulated in cancerous liver cells, carboxyfluorescein (FAM)-labeled aptamers were prepared as fluorescent imaging reagents, and successful differentiation of cancerous liver cells over normal liver cells was achieved, which demonstrated the application feasibility of the selected aptamers. This approach obviated a tedious glycan preparation process and allowed favorable expose of the intrinsic flexible conformation of natural glycans. Therefore, it holds great promise for developing glycan-specific aptamers for challenging applications such as cancer targeting.

We developed a fluorescent aptamer/graphene oxide (GO)-based biosensor to detect sulfamethazine (SMZ) residues in animal-derived foods. The SMZ-bound aptamers were identified and screened with an improved GO-SELEX technique using non-immobilizing ssDNA library. After seven rounds of selection, six SMZ aptamers were sequenced and analyzed for secondary structure, and their affinity and specificity were assessed by binding assays. The truncated aptamer (SMZ1S: 5'-CGTTAGACG-3') with a unique stem-loop structure showed the highest affinity (K d  = 24.6 nM) to SMZ and was used to develop a GO-based fluorescent aptasensor. The binding mechanism between SMZ1S and SMZ was further analyzed by molecular docking. Under optimal conditions, the fluorescent aptasensor showed low detection limits (0.35 ng/mL) and a wide dynamic linear range (from 2 to 100 ng/mL). The aptasensor was also validated against real samples spiked with SMZ, which showed a fluorescence recovery from 93.9 to 108.8% and a coefficient of variation of < 12.7%. Taken together, these results suggest that this novel aptasensor can be used to sensitively, selectively, and accurately detect SMZ residues in foods. Schematic illustration of fluorescent aptasensor based on aptamer/graphene oxide complex detection of of SMZ.

Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.

One of the pivotal steps in aptamer selection is the amplification of target-specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in-depth study of using 5-indolyl-AA-dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo-) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water-in-oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high-throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Exosomes, small extracellular vesicles, are released by various cell types. They are found in bodily fluids, including blood, urine, serum, and saliva, and play essential roles in intercellular communication. Exosomes contain various biomarkers, such as nucleic acids and proteins, that reflect the status of their parent cells. Since they influence tumorigenesis and metastasis in cancer patients, exosomes are excellent noninvasive potential indicators for early cancer detection. Aptamers with specific binding properties have distinct advantages over antibodies, making them effective versatile bioreceptors for the detection of exosome biomarkers. Here, we review various aptamer-based exosome detection approaches based on signaling methods, such as fluorescence, colorimetry, and chemiluminescence, focusing on electrochemical strategies that are easier, cost-effective, and more sensitive than others. Further, we discuss the clinical applications of electrochemical exosome analysis strategies as well as future research directions in this field.

Electrochemical aptamer-based (E-AB) sensors, exploiting binding-induced changes in biomolecular conformation, are rapid, specific, and selective and perform well even in a complex matrix, such as directly in whole blood and even in vivo. However, like all sensors employing biomolecular recognitions, E-AB sensors suffer from an inherent limitation of single-site binding, i.e., its fixed dose-response curve. To circumvent this, we employ here distal-site mutation and allosteric inhibition to rationally tune the dynamic range of E-AB sensors, achieving sets of sensors with a significantly varied target affinity (∼3 orders of magnitude). Using their combination, we recreate several approaches to narrow (down to 5-fold) or extend (up to 2000-fold) the dynamic range of biological receptors. The thermodynamic consequences of aptamer-surface interactions are estimated via the free-energy difference in solution-phase and surface-bound biosensors employing the same aptamer as a recognition element, revealing that an allostery strategy provides a more predictable and efficient means to finely control the target affinity and dynamic range. Such an ability to rationally modulate the affinity of biomolecule receptors would open the door to applications including cancer therapy, bioelectronics, and many other fields employing biomolecule recognition.

Aptamers are chemically synthetic single-stranded DNA or RNA molecules selected by molecular evolution. They have been widely used as attractive tools in biosensing and bioimaging because they can bind to a large variety of targets with high sensitivity and high affinity and specificity. As recognition elements, aptamers contribute in particular to cancer diagnostics by recognizing different cancer biomarkers, while they can also facilitate ultrasensitive detection by further employing signal amplification elements. Optical techniques have been widely used for direct and real-time monitoring of cancer-related biomolecules and bioprocesses due to the high sensitivity, quick response, and simple operation, which has greatly benefited cancer diagnostics. In this review, we highlight recent advances in optical platform-based sensing strategies for cancer diagnostics aided by aptamers. Limitations and current challenges are also discussed.

Human mucin-1 (MUC1) has attracted considerable attention owing to its overexpression in diverse malignancies. Here, for the rapid and efficient detection of MUC1, we present a SERS-colorimetric dual-mode aptasensor, by integrating SERS probes with magnetic separation, which has several distinctive advantages. Using such a dual-mode aptasensor, the colorimetric functionality is distinguishable by the naked eye, providing a fast and straightforward screening ability for the detection of MUC1. Moreover, SERS-based detection greatly improves the detection sensitivity, reaching a limit of detection of 0.1 U/mL. In addition, the combination of SERS and colorimetric method holds the advantages of these two techniques and thereby increases the reliability and efficiency of MUC1 detection. On the one hand, the magnetic nanobeads functionalized with MUC1-specific aptamer were utilized as an efficient capturing substrate for separating MUC1 from biological complex medium. On the other hand, the gold-silver core-shell nanoparticles modified with Raman reporters and the complementary sequences of MUC1 were used as the signal indicator, which could simultaneously report the SERS signal and colorimetric change. This strategy can achieve a good detection range and realize MUC1 analysis in real patients' samples. Thus, we anticipate that this kind of aptasensor would provide promising potential applications in the diagnosis and prognosis of cancers. Graphical abstract.

mRNAs play key roles in regulating diverse cellular functions. In many cases, mRNAs exhibit distinct intracellular localizations that are necessary for the spatiotemporal control of protein expression in cells. Therefore, imaging the localization and dynamics of these mRNAs is crucial for understanding diverse aspects of cellular function. In this review, we summarize how mRNA imaging can be achieved using tethered fluorescent proteins and fluorogenic aptamers. We discuss 'fluorogenic proteins' and describe how these recently developed RNA-regulated fluorescent proteins simplify mRNA imaging experiments., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SRJ reports serving as a scientific founder, advisor to, and owns equity in Lucerna., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The K d values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

In this study, we successfully developed a simple and rapid method for simultaneous detection of Salmonella spp., Listeria monocytogenes , and Escherichia coli using gold nanoparticles and the aptamer aptasensor. We screened 25 specific DNA aptamer candidates against these pathogens using whole-cell S ystematic E volution of L igands by EX ponential enrichment. Among them, Ap6 was selected due to its low energy minimization values of -12.25 and -27.67 kcal/mol derived from MFold and RNAFold analysis, respectively. The assay presented in this study allowed the visual colorimetric detection of labeled colloidal gold nanoparticles as well as determination of UV absorbance at 625 and 525 nm under optimized conditions. The detection limit of this aptasensor was as less as 10 5 CFU/ml. A random investigation of 50 meat samples, including ham and chicken sausages, collected from the local market revealed 96% accuracy, 96% specificity, and 100% sensitivity of the assay. The colorimetric aptasensor can accomplish one-step detection without pre-culture, DNA extraction, and amplification. Hence, it is an easy, rapid, specific, and qualitative assay that can be used as a point-of-care testing to directly detect multiplex foodborne pathogens.

In seeking to develop and optimize reagentless electroanalytical assays, a consideration of the transducing interface features lies key to any subsequent sensitivity and selectivity. This review briefly summarizes some of the most commonly used receptive interfaces that have been employed within the development of impedimetric molecular sensors. We discuss the use of high surface area carbon, nanoparticles, and a range of bioreceptors that can subsequently be integrated. The review spans the most commonly utilized biorecognition elements, such as antibodies, antibody fragments, aptamers, and nucleic acids, and touches on some novel emerging alternatives such as nanofragments, molecularly imprinted polymers, and bacteriophages. Reference is made to the immobilization chemistries available along with a consideration of both optimal packing density and recognition probe orientation. We also discuss assay-relevant mechanistic details and applications in real sample analysis.

Colorimetric platform using the aggregation of gold nanoparticles (AuNPs) is a pretty simple method for biosensing, but advanced instruments such as specterophotometer is still needed to achieve accurately quantitative readout. Aggregated AuNPs exhibit excellent photothermal properties under near-infrared laser irradiation, which is significantly different from non-aggregated AuNPs. Herein, given the different photothermal effect, we translated the AuNPs-based colorimetric assay into a photothermal assay for the quantitative detection of adenosine using a thermometer as readout. Short single-stranded DNA (ssDNA, adenosine aptamer) was adsorbed on the surface of AuNPs and hence prevented the aggregation of AuNPs under high ionic concentration. The presence of adenosine caused the structural change of ssDNA and the AuNPs became aggregated. The enhanced temperature under NIR-laser irradiation has a linear response to the concentration of adenosine in the range of 2.0-50.0 μM. The detection limit was 1.7 μM. This proposed method is portable, easy and applicable to the quantitative assay of other targets by simply replacing of the sequence of ssDNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The chemical composition of cells at the molecular level determines their growth, differentiation, structure, and function. Probing this composition is powerful because it provides invaluable insight into chemical processes inside cells and in certain cases allows disease diagnosis based on molecular profiles. However, many techniques analyze fixed cells or lysates of bulk populations, in which information about dynamics and cellular heterogeneity is lost. Recently, nucleic-acid-based probes have emerged as a promising platform for the detection of a wide variety of intracellular analytes in live cells with single-cell resolution. Recent advances in this field are described and common strategies for probe design, types of targets that can be identified, current limitations, and future directions are discussed., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7-8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Theranostics cover emerging technologies for cell biomarking for disease diagnosis and targeted introduction of drug ingredients to specific malignant sites. Theranostics development has become a significant biomedical research endeavor for effective diagnosis and treatment of diseases, especially cancer. An efficient biomarking and targeted delivery strategy for theranostic applications requires effective molecular coupling of binding ligands with high affinities to specific receptors on the cancer cell surface. Bioaffinity offers a unique mechanism to bind specific target and receptor molecules from a range of non-targets. The binding efficacy depends on the specificity of the affinity ligand toward the target molecule even at low concentrations. Aptamers are fragments of genetic materials, peptides, or oligonucleotides which possess enhanced specificity in targeting desired cell surface receptor molecules. Aptamer-target binding results from several inter-molecular interactions including hydrogen bond formation, aromatic stacking of flat moieties, hydrophobic interaction, electrostatic, and van der Waals interactions. Advancements in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has created the opportunity to artificially generate aptamers that specifically bind to desired cancer and tumor surface receptors with high affinities. This article discusses the potential application of molecular dynamics (MD) simulation to advance aptamer-mediated receptor targeting in targeted cancer therapy. MD simulation offers real-time analysis of the molecular drivers of the aptamer-receptor binding and generate optimal receptor binding conditions for theranostic applications. The article also provides an overview of different cancer types with focus on receptor biomarking and targeted treatment approaches, conventional molecular probes, and aptamers that have been explored for cancer cells targeting., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Precise detection and effective treatment are crucial to prolong cancer patients' lives. Surface-enhanced Raman scattering (SERS) imaging coupled with photothermal therapy has been considered a precise and effective strategy for cancer theranostics. Nevertheless, Raman reporters employed in the literature usually possessed multiple shift peaks in the fingerprint region, which are overlapped with background signals from endogenous biological molecules. Herein, we fabricated a new kind of bioorthogonal Raman reporter and aptamer functionalized SERS nanotags. The SERS nanotags demonstrated a strong Raman signal at 2205 cm -1 in the biologically Raman-silent region and recognized MCF-7 breast cancer cells for Raman imaging with high specificity. Laser irradiation induced serious toxicity of MCF-7 cells due to the excellent photothermal capability of the SERS nanotags. After intravenous administration of the SERS nanotags, tumor Raman spectral detection and mapping in living mice were successfully achieved. Further in vivo antitumor experiments manifested that the aptamer-modified SERS nanotags significantly restrained tumor growth after laser irradiation with 99% inhibition rate and good biocompatibility. These results clearly revealed that the SERS nanotags could serve as a novel and precise theranostic platform for in vivo cancer diagnosis and photothermal therapy.

Vaspin is a protein present in human serum that can cause type-2 diabetes, obesity, and other cardiovascular diseases. We report fluorescent upconverting nanoparticles (UCNPs)-based lateral flow biosensor for ultrasensitive detection of Vaspin. A pair (primary and secondary) of cognate aptamers was used that has duo binding with Vaspin. UCNPs with a diameter of around 100 nm were used as a tag to label a detection probe (secondary aptamer). A primary aptamer (capture probe) was immobilized on the test zone. Sandwich type hybridization reactions among the conjugate probe, target Vaspin, and primary aptamer were performed on the lateral flow biosensor. In the presence of target Vaspin, UCNPs were captured on the test zone of the biosensor and the fluorescent intensity of the captured UCNPs was measured through a colorimetric app under NIR. Fluorescence intensity indicates the quantity of Vaspin present in the sample. A range of Vaspin concentration across 0.1-55 ng ml -1 with a Limit of detection (LOD) 39 pg ml -1 was tested through this UCNPs based LFSA with high sensitivity, reproducibility and repeatability, whereas it's actual range in human blood is from 0.1 to 7 ng ml -1 . Therefore, this research provides a well-suited lateral flow strip with an ultrasensitive and low-cost approach for the early diagnosis of type-2 diabetes and this could be applied to any targets with a duo of aptamers generated., (Copyright © 2019. Published by Elsevier B.V.)

Mycotoxins are toxic compounds produced by the metabolism of certain fungi that threaten the food and agricultural industry. Over hundreds of mycotoxins, one of the most common toxins, zearalenone (ZEN), has toxic effects on human and animal health due to its mutagenicity, treatogenicity, carcinogenicity, nephrotoxicity, immunotoxicity, and genotoxicity. In this work, attenuated internal reflection spectroscopic ellipsometry (AIR-SE) combined with the signal amplification via surface plasmon resonance conditions that were proved to be a highly sensitive analytical tool in bio-sensing was developed for the sensitive and selective ZEN detection in cereal products such as corn, wheat, rice, and oat. Combined with the oligonucleotide aptamer for ZEN recognition, our proposed method showed good performance with yielding 0.08 ng/mL LOD and 0.01-1000 ng/mL detection range. A mini-review was also introduced in, to compare various methods for ZEN detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This review, with 201 references, describes the recent advancement in the application of carbonaceous nanomaterials as highly conductive platforms in electrochemical biosensing. The electrochemical biosensing is described in introduction by classifying biosensors into catalytic-based and affinity-based biosensors and statistically demonstrates the most recent published works in each category. The introduction is followed by sections on electrochemical biosensors configurations and common carbonaceous nanomaterials applied in electrochemical biosensing, including graphene and its derivatives, carbon nanotubes, mesoporous carbon, carbon nanofibers and carbon nanospheres. In the following sections, carbonaceous catalytic-based and affinity-based biosensors are discussed in detail. In the category of catalytic-based biosensors, a comparison between enzymatic biosensors and non-enzymatic electrochemical sensors is carried out. Regarding the affinity-based biosensors, scholarly articles related to biological elements such as antibodies, deoxyribonucleic acids (DNAs) and aptamers are discussed in separate sections. The last section discusses recent advancements in carbonaceous screen-printed electrodes as a growing field in electrochemical biosensing. Tables are presented that give an overview on the diversity of analytes, type of materials and the sensors performance. Ultimately, general considerations, challenges and future perspectives in this field of science are discussed. Recent findings suggest that interests towards 2D nanostructured electrodes based on graphene and its derivatives are still growing in the field of electrochemical biosensing. That is because of their exceptional electrical conductivity, active surface area and more convenient production methods compared to carbon nanotubes. Graphical abstract Schematic representation of carbonaceous nanomaterials used in electrochemical biosensing. The content is classified into non-enzymatic sensors and affinity/ catalytic biosensors. Recent publications are tabulated and compared, considering materials, target, limit of detection and linear range of detection.

Influenza A viruses (IAVs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. IAVs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian IAVs (HPAI). For this study, we infected A549 human lung cells with lab prototype A/PR/8/34 (H1N1) (PR8), a seasonal H1N1 (RV733), the 2009 pandemic H1N1 (pdm09), or with two avian strains, an H5N1 HPAI strain or an H7N9 strain that has low pathogenicity in birds but high pathogenicity in humans. We used a newly-developed aptamer-based multiplexed technique (SOMAscan ® ) to examine >1300 human lung cell proteins affected by the different IAV strains, and identified more than 500 significantly dysregulated cellular proteins. Our analyses indicated that the avian strains induced more profound changes in the A549 global proteome compared to all tested low-pathogenicity H1N1 strains. The PR8 strain induced a general activation, primarily by upregulating many immune molecules, the seasonal RV733 and pdm09 strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems.

A disposable package of biosensors was developed along with the corresponding guidelines for early detection of the acute lymphoblastic leukemia cancer. This proposed cancer-screening package included a DNA sensor and an aptasensor as two main types of biosensors. The biosensors were used simultaneously. This combination of sensors can detect not only the presence of mutant genes but also the biomarkers of cancer. At current work, the combination of sensors were used to detect the presence of BCR-ABL1 as a mutant gene and CEA as a biomarkers of cancer, such a capability makes the package liable for early and certain detection of acute lymphoblastic leukemia. To construct both the DNA sensor and the aptasensor, a nanocomposite consisting of electrosynthesis carbon quantum dots and biosynthesized gold nanoparticles was applied. The construction of these biosensors was characterized using four different electrochemical methods including DPV (Differential Pulse Voltammetry), EIS (Electrochemical Impedance Spectroscopy), CV (Cyclic Voltammetry) and chronoamperometry. The peak current of a catechol solution that was used as an electroactive probe on the biosensor was linearly related to the logarithm of the concentrations of the target DNA and the target antigen in the range of 10 pM to 100 μM and 1 pg mL -1 to 0.001 g mL -1 with the detection limits of 1.5 pM and 0.26 pg mL -1 respectively, which are quite good results., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The detection of alpha-fetoprotein (AFP) is of great importance for hepatocellular carcinoma (HCC) diagnosis, but it needs to be further improved because of poor sensitivity and complicated operating steps. In this paper, a simple and sensitive homogeneous apatasensor for AFP has been developed based on Förster resonance energy transfer (FRET) where the AFP aptamer labeled luminescent CdTe quantum dots (QDs) as a donor and anti-AFP antibody functional gold nanoparticles (AuNPs) as an acceptor. In the presence of AFP, the bio-affinity between aptamer, target, and antibody made the QDs and AuNPs close enough, thus the fluorescence of CdTe QDs quenched though the FRET between QD and AuNP. The fluorescent aptasensor for AFP showed a concentration-dependent decrease of fluorescence intensity in the low nanomolar range and a detecting linear range of 0.5-45 ng mL -1 , with a detection limit of 400 pg mL -1 . Moreover, this homogeneous aptasensor is simple and reliable, and obtained satisfying results for the detection of AFP in human serum samples. With more and more aptamers for biomarkers have been selected gradually, this approach could be easily extended to detection of a wide range of biomarkers. The proposed aptasensor has great potential for carcinoma screening in point-of-care testing and even in field use., (Copyright © 2019. Published by Elsevier B.V.)

To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer-target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.

We propose an in situ and label-free method for detection of biomolecular recognition events by use of a nanochannel-ion channel hybrid device integrated with an electrochemical detector. The aptamer is first immobilized on the outer surface of the nanochannel-ion channel hybrid. Its binding with target thrombin in solution considerably regulates the mass-transfer behavior of the device owing to the varied surface charge density and effective channel size. Via the electrochemical detector, the changed mass-transport property can be monitored in real time, which enables in situ and label-free detection of thrombin-aptamer recognition. The solution pH has a significant influence on detection sensitivity. Under optimal pH conditions, a detection limit as low as 0.22 fM thrombin can be achieved, which is much lower than most reported work. The present nanofluidic device provides a simple, ultrasensitive, and label-free platform for monitoring biomolecular recognition events, which would hold great potential in exploring the functions and reaction mechanisms of biomolecules in living systems.

Aptamers have several positive advantages that made them eminent as a potential factor in diagnosing and treating diseases such as their application in prevention and treatment of diabetes. In this opinion-based mini-review article, we aimed to investigate the DNA and RNA-based hybrid molecules specifically aptamers and had a logical conclusion as a promising future perspective in early diagnosis and treatment of diabetes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

With the advent of the high-throughput technologies and exciting times for biology, the discipline of analytical methodology is experiencing a surge in the growth and the scope. Over the years, multitude of analytical techniques have evolved from a work-intensive, low sensitivity and high volume of reagent and sample consumption endeavor to automated, better selectivity, lower limit of quantification and cost-effective techniques for biological research. In this review, we give an overview of the currently available wide range of cell-based and noncell based and structural based analytical techniques, their principle and biological applications. The analytical techniques discussed in this paper includes surface plasmon resonance, electrophoresis, enzyme linked immunosorbent assay, Western blotting, flow cytometry, fluorescence activated cell sorting, mass spectrometry, nuclear magnetic resonance and x-ray crystallography.