Bioanalytical Applications Research Articles

In-silico analytical chemistry contributions to analytical and bio-analytical applications in spectroscopic and chromatographic techniques: molecular mechanical and quantum insights

In-silico analytical chemistry contributions to analytical and bio-analytical applications in spectroscopic and chromatographic techniques: molecular mechanical and quantum insights

Microarray-Based Electrochemical Biosensing.

Microarrays are widely utilized in bioanalysis. Electrochemical biosensing techniques are often applied in microarray-based assays because of their simplicity, low cost, and high sensitivity. In such systems, the electrodes and sensing elements are arranged in arrays, and the target analytes are detected electrochemically. These sensors can be utilized for high-throughput bioanalysis and the electrochemical imaging of biosamples, including proteins, oligonucleotides, and cells. In this chapter, we summarize recent progress on these topics. We categorize electrochemical biosensing techniques for array detection into four groups: scanning electrochemical microscopy, electrode arrays, electrochemiluminescence, and bipolar electrodes. For each technique, we summarize the key principles and discuss the advantages, disadvantages, and bioanalysis applications. Finally, we present conclusions and perspectives about future directions in this field.

RNA-cleaving DNAzymes for accurate biosensing and gene therapy.

RNA-cleaving DNAzymes, which are single-stranded catalytic DNA, have attracted considerable attention in bioanalysis and biomedical applications because of their high stability, high catalytic activity, easy synthesis, easy functionalization, and modification. By incorporating these DNAzymes with amplification systems, the sensing platforms can be used to detect a series of targets with high sensitivity and selectivity. In addition, these DNAyzmes possess therapeutic potential by cutting the mRNA in cells and viruses to regulate the expression of the corresponding proteins. This review systematically summarizes the applications of RNA-cleaving DNAzymes in recent years, explaining the uniqueness and superiority of RNA-cleaving DNAzymes in biosensing and gene therapy. Finally, this review extends the discussion to the challenges and perspectives of applying RNA-cleaving DNAzyme as a diagnostic and therapeutic agent. This review provides the researchers with valuable suggestions and promotes the development of DNAzymes for accurate analysis, early diagnosis, and effective therapy in medicine and their broader applications beyond biomedicine.

Construction and bioanalytical applications of poly-adenine-mediated gold nanoparticle-based spherical nucleic acids.

Owing to the versatile photophysical and chemical properties, spherical nucleic acids (SNAs) have been widely used in biosensing. However, traditional SNAs are formed by self-assembly of thiolated DNA on the surface of a gold nanoparticle (AuNP), where it is challenging to precisely control the orientation and surface density of DNA. As a new SNA, a polyadenine (polyA)-mediated SNA using the high binding affinity of consecutive adenines to AuNPs shows controllable surface density and configuration of DNA, which can be used to improve the performance of a biosensor. Herein, we first introduce the properties of polyA-mediated SNAs and fundamental principles regarding the polyA-AuNP interaction. Then, we provide an overview of current representative synthesis methods of polyA-mediated SNAs and their advantages and disadvantages. After that, we summarize the application of polyA-mediated SNAs in biosensing based on fluorescence and colorimetric methods, followed by discussion and an outlook of future challenges in this field.

А Method for the Quantitative Determination of the Active Receptor of Beta-lactam Antibiotics BlaR-CTD for Bioanalytical Applications

A sandwich bioassay for the quantitative determination of the recombinant beta-lactam receptor BlaR-CTD possessing ligand binding activity and immunoreactivity has been developed. In the bioassay system, BlaR-CTD present in a biological liquid or standard sample binds via its receptor site to ampicillin immobilized in a microplate well and interacts through the epitopes of its peripheral structure with specific polyclonal antibodies. The analytical sensitivity of the method proved to be 2 ng/mL, and its concentration range was 5–215 ng/mL. In the processes of heterological expression, isolation and reagent forms preparation, the biological activity of BlaR-CTD was monitored and its stability was evaluated. High purity recombinant beta-lactam receptor BlaR-CTD was obtained. The protein was shown to have a sufficiently high resistance to denaturation by chaotropic agents (urea and guanidine hydrochloride), and it was stable over a wide pH range. Also, we proposed the constructions and procedures of competitive bioassays for beta-lactam antibiotics using microplates (analytical sensitivity – 0.02 ng/mL, IC50 = 0.28 ng/mL) or chromatographic test-strips (detection limit 1–2 ng/mL), which are based on the receptor and antigenic properties of BlaR-CTD.

Design and bioanalytical applications of stochastic DNA walkers.

Synthetic DNA walkers that are inspired by the walking behaviors of naturally occurring motor proteins have emerged into an important subfield of DNA nanotechnology. While early DNA walkers were designed to walk on one-dimensional (1D) DNA tracks, the development of DNA origami and DNA functionalized micro-/nanomaterials has enabled diverse 2D and 3D tracks. Random walking becomes possible in such platforms and such stochastic DNA walkers can be engineered with much-improved speed and processivity. The invention and improvement of diverse stochastic DNA walkers have made them ideal amplification platforms for analytical and diagnostic applications. In this feature article, we first review the development of DNA walkers with a historical aspect and then focus on the advances in stochastic DNA walkers. We finally elaborated our research efforts to design varying 3D stochastic DNA walkers for rapid and amplified detection of biologically important nucleic acids and proteins.

Coordination polymer nanospheres based on folic acid: Upconversion properties, cytotoxicity study and drug delivery

Rare earth doped upconversion nanoparticles have been widely studied for their applications in bioanalysis and medical imaging. In this work, rare earth doped coordination polymer nanospheres with folic acid as ligand were prepared via a general and simple solvothermal method. A series of characterization results showed that the nanoparticles were an amorphous hollow nanosphere structure with diameter of ca.150 nm. Under NIR irradiation at 980 nm, the upconversion emission around 663 nm originated from 4F9/2→4I15/2 of Er3+ was successfully realized. In addition, the nanospheres was proved to have good biocompatibility and high loading efficiency for doxorubicin.

A Synergistic Dual-Channel Sensor for Ultrasensitive Detection of Pseudomonas aeruginosa by DNA Nanostructure and G-Quadruplex

Pseudomonas aeruginosa is one of the foodborne pathogenic bacteria that greatly threatens human health. An ultrasensitive technology for P. aeruginosa detection is urgently demanded. Herein, based on the mechanism of aptamer-specific recognition, an electrochemical-colorimetric dual-mode ultrasensitive sensing strategy for P. aeruginosa is proposed. The vertices of DNA tetrahedral nanoprobes (DTNPs), that immobilized on the gold electrode were modified with P. aeruginosa aptamers. Furthermore, the G-quadruplex, which was conjugated with a P. aeruginosa aptamer, was synthesized via rolling circle amplification (RCA). Once P. aeruginosa is captured, a hemin/G-quadruplex, which possesses peroxidase-mimicking activity, will separate from the P. aeruginosa aptamer. Then, the exfoliated hemin/G-quadruplexes are collected for oxidation of the 3,3',5',5'-tetramethylbenzidine for colorimetric sensing. In the electrochemical mode, the hemin/G-quadruplex that is still bound to the aptamer catalyzes polyaniline (PANI) deposition and leads to a measurable electrochemical signal. The colorimetric and electrochemical channels demonstrated a good forward and reverse linear response for P. aeruginosa within the range of 1-108 CFU mL-1, respectively. Overall, compared with a traditional single-mode sensor for P. aeruginosa, the proposed dual-mode sensor featuring self-calibration not only avoids false positive results but also improves accuracy and sensitivity. Furthermore, the consistency of the electrochemical/colorimetric assay was verified in practical meat samples and showed great potential for applications in bioanalysis.

The Roadmap of Graphene-Based Sensors: Electrochemical Methods for Bioanalytical Applications.

Graphene (GR) has engrossed immense research attention as an emerging carbon material owing to its enthralling electrochemical (EC) and physical properties. Herein, we debate the role of GR-based nanomaterials (NMs) in refining EC sensing performance toward bioanalytes detection. Following the introduction, we briefly discuss the GR fabrication, properties, application as electrode materials, the principle of EC sensing system, and the importance of bioanalytes detection in early disease diagnosis. Along with the brief description of GR-derivatives, simulation, and doping, classification of GR-based EC sensors such as cancer biomarkers, neurotransmitters, DNA sensors, immunosensors, and various other bioanalytes detection is provided. The working mechanism of topical GR-based EC sensors, advantages, and real-time analysis of these along with details of analytical merit of figures for EC sensors are discussed. Last, we have concluded the review by providing some suggestions to overcome the existing downsides of GR-based sensors and future outlook. The advancement of electrochemistry, nanotechnology, and point-of-care (POC) devices could offer the next generation of precise, sensitive, and reliable EC sensors.

Ultra-small highly fluorescent zinc-based metal organic framework nanodots for ratiometric visual sensing of tetracycline based on aggregation induced emission

Color tonality by intrinsic fluorescent metal-organic frameworks (MOFs) is highly desirable in bioanalytical applications due to its stability, low-cost and robustness with no need for functionalization and/or encapsulation of fluorophores. In the present work, ultra-small and higly fluorescent zinc-based MOFs (FMOF-5) were synthesized. The prepared FMOFs were around 5 nm in size, and gave strong blue emission at 440 nm when excited at 350 nm. Interestingly, tetracycline (TC) selectively tuned the blue emission of FMOF-5 to greenish-yellow emission (520 nm) with dramatic enhancement through aggregation induced emission (AIE). The fluorimetric analysis of TC was carried out through the ratiometric peak intensities of F520/F440, with detection limit (LOD) of 5 nM. To realize quantitative point-of-care based on color tonality, a smartphone integrated with the ratiometric visual platform was thereby design. Hence, TC was visually detected with LOD of 10 nM. The prepared FMOF-5-based probe showed high stability (3 months) and reusability (∼10 times). The developed visual-based platform presents great promise for practical point of care testing due to its low-cost, robustness, ruggedness, simple operation, and excellent selectivity and repeatability.

Recent Advances in Excimer-Based Fluorescence Probes for Biological Applications

The fluorescent probe is a powerful tool for biological sensing and optical imaging, which can directly display analytes at the molecular level. It provides not only direct visualization of biological structures and processes, but also the capability of drug delivery systems regarding the target therapy. Conventional fluorescent probes are mainly based on monomer emission which has two distinguishing shortcomings in practice: small Stokes shifts and short lifetimes. Compared with monomer-based emission, excimer-based fluorescent probes have large Stokes shifts and long lifetimes which benefit biological applications. Recent progress in excimer-based fluorescent sensors (organic small molecules only) for biological applications are highlighted in this review, including materials and mechanisms as well as their representative applications. The progress suggests that excimer-based fluorescent probes have advantages and potential for bioanalytical applications.

Stimuli‐Responsive RNA‐Cleaving DNAzyme for Biomedical Application

AbstractDNAzymes are synthetic deoxynucleotide oligomers with catalytic properties selected through in vitro systematic evolution of ligands by exponential enrichment (SELEX) technique. RNA‐cleaving DNAzymes have attracted increasing attentions in bioanalysis and biomedical applications due to their simple synthesis and modification, stable structure, and high catalytic activity. Especially, their different modes of activity regulation enable their extensive theranostic development. This review introduces the in vitro selection of DNAzymes and some typical ones, followed by the description of various stimuli‐responsive regulation strategies on DNAzyme activity, including metal ion cofactors, small molecules, protease, light, and heat. Then the recent advances of DNAzyme‐based biosensors, therapeutic applications, cell‐cell interaction regulation, and logic operation are summarized. At last, the current challenges and possible development of DNAzyme‐based biosensors are discussed.

Bioanalytical Applications of Graphene Quantum Dots for Circulating Cell-Free Nucleic Acids: A Review.

Graphene quantum dots (GQDs) are carbonaceous nanodots that are natural crystalline semiconductors and range from 1 to 20 nm. The broad range of applications for GQDs is based on their unique physical and chemical properties. Compared to inorganic quantum dots, GQDs possess numerous advantages, including formidable biocompatibility, low intrinsic toxicity, excellent dispensability, hydrophilicity, and surface grating, thus making them promising materials for nanophotonic applications. Owing to their unique photonic compliant properties, such as superb solubility, robust chemical inertness, large specific surface area, superabundant surface conjugation sites, superior photostability, resistance to photobleaching, and nonblinking, GQDs have emerged as a novel class of probes for the detection of biomolecules and study of their molecular interactions. Here, we present a brief overview of GQDs, their advantages over quantum dots (QDs), various synthesis procedures, and different surface conjugation chemistries for detecting cell-free circulating nucleic acids (CNAs). With the prominent rise of liquid biopsy-based approaches for real-time detection of CNAs, GQDs-based strategies might be a step toward early diagnosis, prognosis, treatment monitoring, and outcome prediction of various non-communicable diseases, including cancers.

Roughness in the periodic potential induces absolute negative mobility in a driven Brownian ratchet.

Absolute negative mobility, where particles move opposite to the direction as governed by the external load, is an anomalous transport property of a Brownian ratchet and has technological implications in mass separation and bioanalytical applications. We numerically investigated here the effect of roughness in symmetric periodic potential on the negative mobility of a driven inertial Brownian ratchet in the presence of an external load. We show that the microscopic spatial heterogeneity of the potential can generate negative mobility which would not otherwise be possible under smooth potential in the concerned parameter space. We determined the optimal condition in terms of parameter space for such anomalous behavior. Our calculations indicate that the shift of balance towards the negative velocity phase in the temporal oscillations of velocity and weakly chaotic dynamics are responsible factors for roughness-induced negative mobility. These calculations highlight a constructive role of roughness in the anomalous transport properties of Brownian ratchet.

Label-free homogeneous photoelectrochemical aptasensing of VEGF165 based on DNA-regulated peroxidase-mimetic activity of metal-organic-frameworks

The application of metal-organic frameworks (MOFs) nanozymes in biosensing has been extensively investigated, however, till now there is still no report on photoelectrochemical (PEC) sensing based on enzyme memetic properties of MOFs. To further expand the utilization of MOFs nanozymes in biosensing, we developed a label-free homogenous PEC aptasensor for the detection of VEGF165, an important cancer biomarker, based on the DNA-regulated peroxidase-mimetic activity of Fe-MIL-88, a type of MOFs. In this strategy, the peroxidase-mimetic property of MOFs is integrated with the label-free homogeneous PEC sensing approach, and highly sensitive detection of VEGF165 is obtained with a detection limit down to 33 fg/mL, superior or comparable to the previously reported values. Moreover, this approach displays outstanding specificity, and has been successfully used to detect VEGF165 added in diluted serum samples. As far as we know, it is the first example to employ the peroxidase-like activity of MOFs in PEC biosensing, which may find potential application in bioanalysis and early disease diagnosis.

Nanozyme Based on Dispersion of Hemin by Graphene Quantum Dots for Colorimetric Detection of Glutathione.

Compared with natural enzymes, nanozymes have the advantages of good catalytic performance, high stability, low cost, and can be used under extreme conditions. Preparation of highly active nanozymes through simple methods and their application in bioanalysis is highly desirable. In this work, a nanozyme based on dispersion of hemin by graphene quantum dot (GQD) is demonstrated, which enables colorimetric detection of glutathione (GSH). GQD was prepared by a one-step hydrothermal synthesis method. Hemin, the catalytic center of heme protein but with low solubility and easy aggregation that limits its catalytic activity, can be dispersed with GQD by simple sonication. The as-prepared Hemin/GQD nanocomplex had excellent peroxidase-like activity and can be applied as a nanozyme. In comparison with natural horseradish peroxidase (HRP), Hemin/GQD nanozyme exhibited a clearly reduced Michaelis–Menten constant (Km) when tetramethylbenzidine (TMB) was used as the substrate. With H2O2 being the substrate, Hemin/GQD nanozyme exhibited a higher maximum reaction rate (Vmax) than HRP. The mechanisms underlying the nanozyme activity were investigated through a free radical trapping experiment. A colorimetric platform capable of sensitive detection of GSH was developed as the proof-of-concept demonstration. The linear detection range was from 1 μM to 50 μM with a low limit of detection of 200 nM (S/N = 3). Determination of GSH in serum samples was also achieved.

Fluorescent and Phosphorescent Nitrogen-Containing Heterocycles and Crown Ethers: Biological and Pharmaceutical Applications.

Fluorescent molecules absorb photons of specific wavelengths and emit a longer wavelength photon within nanoseconds. Recently, fluorescent materials have been widely used in the life and material sciences. Fluorescently labelled heterocyclic compounds are useful in bioanalytical applications, including in vivo imaging, high throughput screening, diagnostics, and light-emitting diodes. These compounds have various therapeutic properties, including antifungal, antitumor, antimalarial, anti-inflammatory, and analgesic activities. Different neutral fluorescent markers containing nitrogen heterocycles (quinolones, azafluoranthenes, pyrazoloquinolines, etc.) have several electrochemical, biological, and nonlinear optic applications. Photodynamic therapy (PDT), which destroys tumors and keeps normal tissues safe, works in the presence of molecular oxygen with light and a photosensitizing drugs (dye) to obtain a therapeutic effect. These compounds can potentially be effective templates for producing devices used in biological research. Blending crown compounds with fluorescent residues to create sensors has been frequently investigated. Florescent heterocyclic compounds (crown ether) increase metal solubility in non-aqueous fluids, broadening the application window. Fluorescent supramolecular polymers have widespread use in fluorescent materials, fluorescence probing, data storage, bio-imaging, drug administration, reproduction, biocatalysis, and cancer treatment. The employment of fluorophores, including organic chromophores and crown ethers, which have high selectivity, sensitivity, and stability constants, opens up new avenues for research. Fluorescent organic compounds are gaining importance in the biological world daily because of their diverse functionality with remarkable structural features and positive properties in the fields of medicine, photochemistry, and spectroscopy.

Paper-based microfluidics: What can we expect?

Paper-based microfluidics: What can we expect?

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

Recent molecularly imprinted polymers applications in bioanalysis.

Molecular imprinted polymers (MIPs) as extraordinary compounds with unique features have presented a wide range of applications and benefits to researchers. In particular when used as a sorbent in sample preparation methods for the analysis of biological samples and complex matrices. Its application in the extraction of medicinal species has attracted much attention and a growing interest. This review focus on articles and research that deals with the application of MIPs in the analysis of components such as biomarkers, drugs, hormones, blockers and inhibitors, especially in biological matrices. The studies based on MIP applications in bioanalysis and the deployment of MIPs in high-throughput settings and optimization of extraction methods are presented. A review of more than 200 articles and research works clearly shows that the superiority of MIP techniques lies in high accuracy, reproducibility, sensitivity, speed and cost effectiveness which make them suitable for clinical usage. Furthermore, this review present MIP-based extraction techniques and MIP-biosensors which are categorized on their classes based on common properties of target components. Extraction methods, studied sample matrices, target analytes, analytical techniques and their results for each study are described. Investigations indicate satisfactory results using MIP-based bioanalysis. According to the increasing number of studies on method development over the last decade, the use of MIPs in bioanalysis is growing and will further expand the scope of MIP applications for less studied samples and analytes.