Exploration of the Interaction of Cadmium and Aptamer by Molecular Simulation and Development of Sensitive Capillary Zone Electrophoresis-Based Aptasensor.

The presence of cadmium ions (Cd2+) in environmental samples demands a fast, sensitive, and selective analytical method that can measure toxic levels. Biosensors based on aptamers (aptasensors) have been developed, but some of them suffer from poor sensitivity and specificity due to the immobilization of aptamers. Here, we employed circular dichroism, molecular docking, and molecular dynamics simulation to reveal that the aptamer gradually undergoes significant conformational changes upon Cd2+ binding. This fact highlights the advantages of biosensors based on free aptamers. So, keeping these results, an analytical method was established for the detection of Cd2+ by utilizing capillary zone electrophoresis (CZE), which is adapted for the free aptamer. So, CZE equipped with aptamer as a detection probe can detect Cd2+ within 4 min in the range from 5 to 250 nM with R2 = 0.994, limit of detection 5 nM (signal-to-noise ratio = 3), and recovery from 92.6 ± 1.6 to 107.4 ± 1.0% in river water samples. Furthermore, the detected concentration in water samples is below the harmful levels (267 nM) recommended by World Health Organization standards in drinking water. This method displays a high sensitivity and specificity for Cd2+. It is found to be superior to existing methods, which use immobilized aptamers, and can be readily expanded to design aptasensors for other targets.

Aptamer-linked photonic crystal hydrogel sensor for rapid point-of-care detection of human immuno-deficiency virus-1 (HIV-1).

The detection of the human immunodeficiency virus-1 (HIV) at an early stage is vital and could be realized through its cell surface glycoprotein-120 (gp120) without virus preprocessing. Here, we present an ssDNA-aptamer-linked photonic crystal (APC) hydrogel sensor for HIV detection which is comprised of photonic crystals (PCs) made of polystyrene nanoparticles embedded in the polyacrylamide hydrogel. ssDNA aptamers specific for gp120 are crosslinked in the hydrogel which can selectively bind to gp120 by hydrogen bonding increasing the PCs particle spacing and swelling of the hydrogel. The binding response can be visually monitored as a color change due to the diffraction of light from PCs and can eventually be measured (1-1000 ng mL-1 of gp120) and 100 to 108 VP mL-1 of HIV by the Debye's ring diameter or a UV/Vis spectrometer. APC-hydrogel can be regenerated by Tris-HCl and EDTA washing buffer system. The sensor demonstrates LOD of 7.1 ±â€¯1.55 ng mL-1 for gp120 and 4 VP mL-1 for the whole HIV, a rapid response of 5 min, reusability up to 70 % (in fifth use), and recovery of 95.4 ±â€¯0.1 % to 99.0 ±â€¯0.2 % in plasma samples. The sensor is cost-effect and stable compared to antibody-based sensors and can be utilized to develop point-of-care testing (POCT) devices for HIV diagnosis.

Aptamer-Linked Photonic Crystal Assay for High-Throughput Screening of HIV and SARS-CoV-2.

We present a microplate assay for the detection of HIV and SARS-CoV-2 which involves the preadsorption of carboxy-modified polystyrene microspheres to the microplate wells and their self-assembly leading to the formation of a photonic crystal colloidal array (PCCA). PCCA is then cross-linked with amino-modified aptamers selected for viral cell surface glycoproteins, i.e., S1-protein of SARS-CoV-2 and gp120 of the human immunodeficiency virus (HIV), to develop an aptamer-linked photonic crystal assay (ALPA). ALPA is then utilized as a proof-of-concept method for the detection of S1-protein, gp120, and two whole viruses, i.e., SARS-CoV-2 and HIV, as well. The aptamers are stable at room temperature and can bind with the viruses' proteins via hydrogen bonding. This binding leads to color generation from PCCA, and the signal can easily be measured and quantified by a UV/vis spectrometer. The assay carries the advantage of a two-step detection process by the addition of the virus sample directly to a 96-well microplate and incubation of 5 min followed by convenient detection through a UV/vis-spectrometer. The assay does not require any additional reagents and can be customized for similar viruses utilizing specific aptamers targeting their cell surface receptors.

Consensus Receptor-Binding Domain-Targeted Aptamer Selection and Designing of a Photonic Crystal-Decorated Aptasensor for SARS-CoV-2.

The frequent emergence of variants of concern (VOC) of SARS-CoV-2 necessitates a sensitive and all-inclusive detection platform that remains viable despite the virus mutations. In this context, we targeted the receptor-binding domain (RBD) of glycoprotein (S-protein) of all VOC and constructed a consensus RBD (cRBD) based on the conserved amino acids. Then, we selected a high-affinity ssDNA novel aptamer specific for the cRBD by an in silico approach. The selected aptamer is utilized to fabricate a photonic crystal (PC)-decorated aptasensor (APC-sensor), which consists of polystyrene nanoparticles polymerized within a polyacrylamide hydrogel. cRBD-responsive ssDNA aptamers are crosslinked in the hydrogel network, which selectively bind to the cRBD and SARS-CoV-2 in saliva samples. The binding response can be visually monitored by swelling of the hydrogel and color generation by diffraction of light from PCs and can be quantified by the diffraction ring diameter or a spectrometer. The sensor delivers a LOD of 12.7 ± 0.55 ng mL-1 for the cRBD and 3 ± 18.8 cells mL-1 for SARS-CoV-2 in saliva samples, with a rapid response of 5 min. The sensor can be stored and regenerated without loss of activity. It can be utilized as a point-of-care testing (POCT) for SARS-CoV-2 diagnosis.

Determination of glycated albumin in serum and saliva by capillary electrophoresis utilizing affinity of 3-acrylamido phenylboronic acid selected by virtual screening and molecular docking.

The glycated albumin (G-alb) is a potential marker of hyperglycemia in diabetes and other neurodegenerative disorders in humans. G-alb's presence in the total human serum albumin (tHSA) is an important indicator in the timely diagnosis of disease. To identify G-alb content, it needs to be isolated from non-glycated albumin (NG-alb). Here, we present Capillary electrophoresis (CE) methods with 3-acrylamido phenylboronic acid (3-APBA) as an entrapped ligand in the agarose gel to develop agarose-3-APBA functional capillary and as an affinity ligand added to the buffer without agarose. 3-APBA was selected by computational virtual screening of several phenylboronic acid (PBA) compounds and other ligands to bind G-alb and separate from NG-alb selectively. The agarose-3-APBA functional capillary method involved agarose gel dilution approach coupled with injection pressure to obtain reduced viscosity and sufficient injection volume of protein samples. The method delivered separation in 9.7 min, with a resolution of 3.4, G-alb recovery up to 65%, and took 25 min to complete the entire process. The second method involved 3-APBA as an affinity ligand in the buffer and delivered separation in 4.2 min, with a resolution of 6.4, G-alb recovery up to 102% recovery, with relatively easy procedures. Therefore, it was further applied to determine G-alb content from tHSA in human serum and saliva. The G-alb found content in serum samples was in the range of 21. 1 ± ± 1.4% to 40.5  ± 1.6% out of tHSA and 25.1  ± 1.6% to 33.3 1.4% in saliva. The binding mechanisms were investigated by molecular dockings, which revealed hydrogen bonding, π-π, and van der walls interactions between 3-APBA and G-alb. The affinity was validated by affinity capillary electrophoresis (ACE), which revealed relatively strong interactions between 3-APBA and G-alb with the binding constant (Kb) of 4.53 × 109M - 1 to the 3.41 × 108M - 1 of 3-APBA and NG-alb. The affinity of 3-APBA toward G-alb was increased at pH 9.0 of the borax-borate (BB) buffer as background electrolyte (BGE). The limit of detection (LOD) was 10 nM, repeatability (RSD, n = 3) ≤ 1.4%, and recovery rate was 87.8 ± 1.6 to 100 ± 1.4% in serum and 97.3 ± 1.3 to 102.6 ± 1.1% in saliva. The sensitivity and reproducibility of the method met the detection requirements.

Glycated albumin based photonic crystal sensors for detection of lipopolysaccharides and discrimination of Gram-negative bacteria.

We present two types of two-dimensional (2D) photonic crystals (PC) hydrogel sensors based on glycated albumin (G-alb) as a proof-of-concept for utilizing recognition between G-alb and bacterial cell surface lipopolysaccharides (LPS) to detect and discriminate Gram-negative bacteria. The G-alb functionalized PC-G-alb hydrogel provides recognition of different LPS via hydrogen bonding and can discriminate different Gram-negative bacteria based on their LPS types. The hydrogel delivered LOD of 0.87 ng mL-1 for E.coli LPS, 153 CFU mL-1 for E.coli, 1.22 ng mL-1 for P.aeruginosa LPS and 225 CFU mL-1 for P.aeruginosa. On the other hand, LPS bioimprinted hydrogel (PC-G-alb-LPSimp) provides selective recognition of E.coli LPS with LOD 0.76 ng mL-1 and for E.coli 58 CFU mL-1, via generation of flexible specific cavities for E.coli and its LPS. The two hydrogels showed remarkable recoveries for both LPS and Gram-negative bacteria in the relevant samples of milk, orange juice, river water, and serum with a short response time of 6-12 min. In the binding process, the hydrogels shrink, and 2D PC particle spacing decreases with diffraction shift from green to blue. The diffraction shifts can be visually observed, measured through Debye's diffraction ring diameter by a laser pointer or determined from a spectrometer.

Determination of Kynurenine Enantiomers by Alpha-Cyclodextrin, Cationic-ßeta-Cyclodextrin and Their Synergy Complemented with Stacking Enrichment in Capillary Electrophoresis.

We present high resolution fast, cost-effective and sensitive Capillary zone electrophoresis (CZE) methods for determination of enantiomeric compounds of Kynurenine pathway, i.e. D, L-Kynurenine (KYN), in human serum and urine samples by cationic-ß-CD and its synergistic dual chiral selector system (SD-CSs) with α-CD in 50 mM borax borate buffer (pH 9.0) as BGE. Acid-mediated stacking enrichment by HCl delivered 15 nM limit of detection (LOD) and 50 nM limit of quantification (LOQ). The methods gave advantages of linearity in the concentration range of 50 nM-1000 nM, reproducibility (RSD ≤ 3.35), selectivity against interfering amino acids, and remarkable recoveries. SD-CSs delivered resolution of D, L-KYN twice that of individual chiral selectors (CSs) under similar conditions. The binding constants (Kb) and electrophoretic mobilities (µeff) of D, L-KYN with different concentrations of CSs were calculated to find the migration order of enantiomers. The chiral recognition mechanism was investigated by molecular docking and molecular mechanics, which revealed strong hydrogen bonding between Kynurenine enantiomers and the SD-CSs as compared to individual CS as the key player in binding, formation of stable complexes which led to the ultimate separation.

Development of Molecularly Imprinted 2D Photonic Crystal Hydrogel Sensor for Detection of L-Kynurenine in Human Serum.

l-Kynurenine (KYN) is a metabolite of the Kynurenine pathway and is a known potential marker of immune suppressant disorders and cancer. Here, we present a molecularly imprinted two dimensional (2D) Photonic crystal (PC) hydrogel sensor for the detection of L-KYN in human serum. The sensor utilizes polystyrene-based 2D PC colloidal arrays (2D PCCA) hydrogel with methacrylic acid as the functional monomer which can imprint the L-KYN template by hydrogen bonding. After removal of the template, the resulting nanocavities in the hydrogel can selectively bind and recognize L-KYN in the serum samples. The binding is selective for L-KYN, which is revealed by shrinkage of the hydrogel volume and decrease in the particle spacing that can be easily monitored through changes in the Debye diffraction ring diameter using a LASER pointer. The sensor demonstrates visible red to green color shift upon binding to L-KYN. The 2D PC sensor demonstrates the limit of detection (LOD) of 50  nM, linear relationship of particle spacing versus L-KYN concentration range (50-1000  nM) with the analytical recovery of up to 92 % in the spiked serum samples. The sensor can distinguish between L-KYN and D-KYN and is re-usable up to five times. The sensor is available for the rapid and quantitative detection of L-KYN in the human serum.

Online reaction based single-step CE for Protein-ssDNA complex obtainment to assist aptamer selection.

CE application in aptamer selection (CE-SELEX) shows more advantages than other selection methods. In this study, an online reaction based single-step CE (ssCE) mode was employed for fast obtaining protein-ssDNA complex. Using human thrombin (H-Thr) and its aptamer Apt29 as models, we accomplished the procedures of mixing, reaction, separation, detection and complex collection in single step online process, which took about 10 min to obtain the H-Thr/Apt29 complex. Important factors, affecting the aptamer and H-Thr interaction (buffer, ratio of aptamer and H-Thr amount), and complex separation and collection (voltage and temperature) were discussed. Later, the online reaction of H-Thr with an 80 nt ssDNA library was realized under optimized conditions, and the H-Thr/ssDNA complex was collected and subjected to PCR. By analyzing the PCR product through capillary gel electrophoresis, the resulting approximative 80 nt DNA length validated the ssDNA sequence in complex. To confirm the availability of ssCE mode, two ssDNA libraries with different lengths (56 nt and 82 nt ssDNA) and three proteins (platelet derived growth factor, PDGF-BB; lactoferrin protein, LF; and single-strand DNA binding protein, SSB) were utilized. Their complex peaks were also observed in electropherograms as expected. Additionally, the online incubation of ssDNA and H-Thr was achieved by stopping the separation voltage for some time when ssDNA passed the H-Thr zone. Our results show the ssCE mode has apparent merits of saving time and sample cost for aptamer selection against protein targets.