Single-Molecule Evaluation of the SARS-CoV-2 Nucleocapsid Protein Using Gold Particle-in-a-Frame Nanostructures Enhanced Fluorescent Assay.

Ultrasensitive evaluation of low-abundance analytes, particularly with limits approaching a single molecule, is a key challenge in the design of an assay for profiling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen. Herein, we report an aptamer claw strategy for directly evaluating the SARS-CoV-2 antigen based on gold particle-in-a-frame nanostructures (Au PIAFs). Au PIAF was used as a metal-enhanced fluorescence material. The assay integrated with a microplate reader achieved a sensitivity of 44 fg·mL-1 in under 3 min and accurately detected the SARS-CoV-2 nucleocapsid protein (N protein) in human saliva samples. When our assay is combined with a single-molecule counting platform, the limit of detection can be as low as 0.84 ag·mL-1. This rapid and ultrasensitive assay holds promise as a tool for screening SARS-CoV-2 and other contagious viruses.

A Rapid SARS-CoV-2 Nucleocapsid Protein Profiling Assay with High Sensitivity Comparable to Nucleic Acid Detection.

Existing nucleic acid and antigen profiling methods for COVID-19 diagnosis fail to simultaneously meet the demands in sensitivity and detection speed, hampering them from being a comprehensive way for epidemic prevention and control. Thus, effective screening of COVID-19 requires a simple, fast, and sensitive method. Here, we report a rapid assay for ultrasensitive and highly specific profiling of COVID-19 associated antigen. The assay is based on a binding-induced DNA assembly on a nanoparticle scaffold that acts by fluorescence translation. By binding two aptamers to a target protein, the protein brings the DNA regions into close proximity, forming closed-loop conformation and resulting in the formation of the fluorescence translator. Using this assay, saliva nucleocapsid protein (N protein) has been profiled quantitatively by converting the N protein molecule information into a fluorescence signal. The fluorescence intensity is enhanced with increasing N protein concentration caused by the metal enhanced fluorescence using a simple, specific, and fast profiling assay within 3 min. On this basis, the assay enables a high recognition ratio and a limit of detection down to 150 fg mL-1. It is 1-2 orders of magnitude lower than existing commercial antigen ELISA kits, which is comparative to or superior than the PCR based nucleic acid testing. Owing to its rapidity, ultrasensitivity, as well as easy operation, it holds great promise as a tool for screening of COVID-19 and other epidemics such as monkey pox.

Aptamer-engineered gold nanorod driven an absorbance enhanced strategy for sensitive biomacromolecule profiling.

Gold nanorods (AuNRs)-based plasmonic biosensor offers new opportunity for quantification of biomacromolecules due to their high designability and low technical demands. However, existing methods for the optical detection of biomacromolecule require the targets to induce the aggregation or etching of AuNRs. This limits the range of targets that can be detected, because molecules at extremely low concentration are difficult to arouse aggregation or etching of AuNRs. Thus, it is still challenge to design a scheme for the biomacromolecules at extremely low concentration which can't arouse aggregation or etching of AuNRs based on their plasmonic property. This study proposes a universal absorbance enhanced strategy for biomacromolecule detection with aptamers engineered AuNRs. The biosensor assay (Apts/AuNRs) is designed through assembly of two aptamers on AuNRs to specified recognize the target biomacromolecules, forming closed-loop conformation based on the proximity-dependent ligation, producing absorbance enhancement in the plasmonic peak of AuNRs. It is interesting that the absorbance enhancement increases gradually with increasing protein concentration within a certain range, whereas no aggregation or etching of AuNRs was observed compared with the typical AuNRs based LSPR sensor. Taking advantage of the excellent near infrared light absorption of AuNRs, Apts/AuNRs could be utilized to detect red protein such as cytochrome C, which exhibited better performance than AuNPs based plasmonic sensor. On this basis, the selectivity detection of cytochrome C with the detection of limit down to picomole level was demonstrated. By changing the type of aptamers on AuNRs, the sensitive and credible method was also utilized for the analysis of telomerase activity in nerve cell lysate. Telomerase activity in 4 × 104 neuroblastoma cell was determined to be about 3.575 U/L, which was close to the result of ELISA kit. Good recovery was achieved using standard samples recovery. This study broadens the scope of AuNRs based plasmonic ‬‬‬‬‬‬property ‬‬‬‬and offer a simple, sensitive and selective strategy for biomacromolecules detection in complexed biofluid.

Low-Fouling Nanoporous Conductive Polymer-Coated Microelectrode for In Vivo Monitoring of Dopamine in the Rat Brain.

In vivo electrochemistry with a carbon-fiber electrode (CFE) is the most useful method for tracking neurochemicals in specific brain regions due to its high spatiotemporal resolution. However, CFE is inevitably subject to surface biofouling that leads to a decrease in sensitivity. Here, we develop a polytannic acid (PTA)-doped nanoporous conductive polyaniline (PANI) membrane-coated CFE to minimize biofouling-induced negative effects for in vivo analysis. The as-prepared PTA-PANI-coated CFE shows excellent antifouling property and enrichment capacity toward electrochemical measurement of dopamine (DA) in physiological pH. The PTA-PANI-coated CFE can in vivo monitor the release of DA induced by electrical stimulation and exhibits almost the same sensitivity in the postcalibration (Spost) and the precalibration (Spre; Spost/Spre = 0.90). We believe this conductive nanoporous membrane-coated CFE offers a new platform for in vivo measurement, which would help probe brain chemistry.

DNA-nanohydrogel self-assembled gold nanoparticles: co-profiling of multiple small molecule reductants in rat brain.

DNA nanohydrogel assembled AuNPs were proposed as a high-throughput multidimensional sensing strategy for small molecule reductant profiling in rat brain. The equilibrium among AuNPs, DNA nanohydrogel and targets produced a unique fingerprint-like pattern for differentiating the reducing capacity.

Collision of Aptamer/Pt Nanoparticles Enables Label-Free Amperometric Detection of Protein in Rat Brain.

Single particle collision is emerging as a powerful and sensitive technique for analyzing small molecules, however, its application in biomacromolecules detection, for example, protein, in complex biological environments is still challenging. Here, we present the first demonstration on the single particle collision that can be developed for the detection of platelet-derived growth factor (PDGF), an important protein involved in the central nervous system in living rat brain. The system features Pt nanoparticles (PtNPs) conjugated with the PDGF recognition aptamer, suppressing the electrocatalytic collision of PtNPs toward the oxidation of hydrazine. In the presence of PDGF, the stronger binding between targeted protein and the aptamer disrupts the aptamer/PtNPs conjugates, recovering the electrocatalytic performance of PtNPs, and allowing quantitative, selective, and highly sensitive detection of PDGF in cerebrospinal fluid of rat brain.

Nanoskiving fabrication of size-controlled Au nanowire electrodes for electroanalysis.

Nanoskiving, benefiting from its simple operation and high reproducibility, is a promising method to fabricate nanometer-size electrodes. In this work, we report the fabrication of Au nanowire electrodes with different shapes and well-controlled sizes through nanoskiving. Au nanowire block electrodes, membrane electrodes and tip electrodes are prepared with good reproducibility. Steady-state cyclic voltammograms (CVs) demonstrate that all these electrodes behave well as nanoband ultramicroelectrodes. A fast heterogeneous electron transfer rate constant can be extracted reliably from steady-state CVs at various size Au nanowire block electrodes by the Koutecký-Levich (K-L) method. The Au nanowire membrane electrodes demonstrate good sensitivity toward the oxidation of catecholamine and could monitor catecholamine released from rat adrenal chromaffin cells stimulated by high K+.

Absorbance enhancement of aptamers/GNP enables sensitive protein detection in rat brains.

An absorbance enhanced probe based on gold nanoparticles (GNPs) was proposed for a protein assay in the cerebrospinal fluid of a rat brain. The GNPs, assembled with two aptamers by proximity ligation, have high anti-salt properties, and good selectivity and response toward proteins, such as interferon-gamma, in the brain.

Multidimensional colorimetric sensor array for discrimination of proteins.

An extensible multidimensional colorimetric sensor array for the detection of protein is developed based on DNA functionalized gold nanoparticles (DNA-AuNPs) as receptors. In the presence of different proteins, the aggregation behavior of DNA-AuNPs was regulated by the high concentrations of salt and caused different color change; while DNA-AuNPs grew induced by the reduction of HAuCl4 and NH2OH as a reductant on the surface of nanoparticles exhibited different morphologies and color appearance for different proteins. The transducers based on AuNPs modified by specific and nonspecific DNA enables naked-eye discrimination of the target analytes. This extensible sensing platform with only two receptors could simultaneously discriminate ten native proteins and their thermally denatured conformations using hierarchical cluster analysis (HCA) at the concentration of 50nM with 100% accuracy. This opens up the possibility of the sensor array to investigate the different conformational changes of biomacromolecules, and it gives a new direction of developing multidimensional transduction principles based on plasmonic nanoparticle conjugates. Furthermore, the sensing system could discriminate proteins at the concentration of 500nM in the presence of 50% human urine, which indicated this sensor array has great potential ability in analyzing real biological fluids. In addition, the multidimensional colorimetric sensor array is suitable for analysis of target analytes in the resource-restricted regions because of rapid, simple, low cost, and in-field detection with the naked eye.

A nanoplasmonic probe as a triple channel colorimetric sensor array for protein discrimination.

The salt-induced aggregation, nanoparticle regrowth and self-assembly behaviors of gold nanoparticles (AuNPs) and DNA conjugates could be changed after interaction with different proteins, generating various color changes and a unique fingerprint pattern for each protein. The triple-channel colorimetric signals have been employed for protein discrimination with the naked eye.

Ratiometric fluorescence sensor arrays based on quantum dots for detection of proteins.

Optical cross-reactive sensor arrays have recently been demonstrated as a powerful tool for high-throughput protein analysis. Nevertheless, applying this technology to protein detection is complicated by many external factors, such as the interfering substances, the background noise, and sample environmental changes in the biological matrix. Herein we demonstrate that a ratiometric fluorescence sensor array based on quantum dots can be employed to circumvent these limitations. Several intrinsic dual-emitting Mn-doped quantum dots capped with different organic functional groups were designed as sensing elements. Distinct and reproducible response patterns against the ratiometric sensor array were obtained from ten proteins in a buffer of different pH (pH 5.7, 7.4, and 8.3) and spiked into human urine. Linear discrimination analysis of the response patterns showed successful differentiation of the analytes at concentrations as low as 50 nM with high identification accuracy. Furthermore, this sensor system also enables the detection of these eight proteins (at 500 nM) in human urine without any treatment. The ratiometric fluorescence change from quantum dots for analysis of proteins can eliminate effectively the signal interference from the pH value change and the fluorescent background in human urine. The present study will open a new avenue to improve the discrimination ability of sensor arrays.

Multidimensional sensor for pattern recognition of proteins based on DNA-gold nanoparticles conjugates.

We presented an extensible multidimensional sensor with conjugated nonspecific dye-labeled DNA sequences absorbed onto gold nanoparticles (DNA-AuNPs) as receptors. At the presence of target protein, DNA was removed from the surface of AuNPs due to the competitive binding, which resulted in a red-to-blue color change along with salt-induced aggregation of AuNPs for colorimetric analysis and fluorescent "turn-on" signal of the labeled dye for fluorescence analysis. The orthogonal and complementary fluorescent and colorimetric signals obtained from each protein were applied to distinguish different proteins. By simply changing the DNA sequences, more dual-channel sensing elements could be easily obtained and added into this multidimensional sensor. This enhanced its discriminating power to the proteins. With three sensing elements, our extensible multidimensional sensing platform exhibited excellent discrimination ability. Eleven proteins at the concentration of 50 nM had been classified with accuracies of 100% by using linear discriminant analysis (LDA). Remarkably, two similar proteins [bovine serum albumin (BSA) and human serum albumin (HSA)] at various concentrations and the mixture of these two proteins with different molar ratios had been successfully discriminated in one LDA plot as well. Furthermore, in the presence of human urine sample, 10 proteins at 1.0 µM could also be well-discriminated. The accuracy of discrimination of unknown samples was all 100% for these experiments. This strategy is a complement of the multidimensional sensing system and traditional sensor platform, offering a new way to develop sensitive array sensing systems.

Dopamine from cirrhotic liver contributes to the impaired learning and memory ability of hippocampus in minimal hepatic encephalopathy.

Background Defective learning/memory ability is a feature of MHE. However, the exact pathophysiological mechanisms leading to the impairment of learning/memory ability in MHE remain not clearly understood. Methods MHE rat modeling by intraperitoneal injection of TAA was successfully established using a Morris water maze, BAEP, and EEG tests. COMT inhibitor, a protein involved in the accumulation of dopamine (DA), was found to be up-regulated in cirrhotic livers in MHE by 2-DE/MS. Results The levels of DA in cirrhotic livers, serums and hippocampuses in the MHE group were more significantly increased than in the control group. In the hippocampuses of MHE rats, NMDA-induced formation of cGMP was reduced by 40 % as determined by in vivo brain microdialysis. Activation of sGC by NO was reduced by 38 %. The expression of NMDAR1, CaM, nNOS and sGC in the hippocampus in the MHE group were more significantly decreased than in controls. Chronic exposure of cultured hippocampus neurons to DA (50 µM) reduced by 53 % the NMDA-induced formation of cGMP. Activation of sGC by NO in these neurons was reduced by 44 %. Down-regulated NMDAR1, CaM, nNOS and sGC were also detected in neurons treated with dopamine, in contrast with the controls. Conclusions This study suggests that when the glutamate-NO-cGMP pathway in the hippocampus is inhibited by the elevation of DA from cirrhotic livers, this in turn may lead to the impairment of learning and memory ability of MHE.