Developing magnetic barcode bead fluorescence assay for high throughput analyzing humoral responses against multiple SARS-CoV-2 variants.

The continuous mutation of SARS-CoV-2 highlights the need for rapid, cost-effective, and high-throughput detection methods. To better analyze the antibody levels against SARS-CoV-2 and its variants in vaccinated or infected subjects, we developed a multiplex detection named Barcode Bead Fluorescence (BBF) assay. These barcode beads were magnetic, characterized by 2-dimensional edges, highly multiplexed, and could be decrypted with visible light. We conjugated 12 magnetic barcode beads with corresponding nine spike proteins (wild-type, alpha, beta, gamma, delta, and current omicrons), two nucleocapsid proteins (wild-type and omicron), and one negative control. First, the conjugated beads underwent serial quality controls via fluorescence labeling, e.g., reproducibility (R square = 0.99) and detection limits (119 pg via anti-spike antibody). Next, we investigated serums from vaccinated subjects and COVID-19 patients for clinical applications. A significant reduction of antibody levels against all variant beads was observed in both vaccinated and COVID-19 studies. Subjects with two doses of mRNA-1273 exhibited the highest level of antibodies against all spike variants compared to two doses of AZD1222 and unvaccinated. We also found that COVID-19 patients showed higher antibody levels against spike beads from wild-type, alpha, beta, and delta. Finally, the nucleocapsid beads served as markers to distinguish infections from vaccinated subjects. Overall, this study developed the BBF assay for analyzing humoral immune responses, which has the advantages of robustness, automation, scalability, and cost-effectiveness.

In situ study of EDC/NHS immobilization on gold surface based on attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS).

In this study, the time-dependent reaction between 11-mercaptoundecanoic acid (11-MUA) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) is precisely characterized using surface enhanced infrared absorption spectroscopy (SEIRAS). According to the high correlation between the spectral results of SEIRAS and the electrochemical behavior, it strongly demonstrates that the EDC/NHS reaction would be obviously interfered by phosphate ions in the neutral pH condition (pH = 7.0).

Aptamer-based sensor for quantitative detection of mercury (II) ions by attenuated total reflection surface enhanced infrared absorption spectroscopy.

A sensing platform based on the attenuated total reflection surface enhanced infrared absorption spectroscopy (ATR-SEIRAS) technique and immobilized aptamer has been proposed herein for the selective detection of mercury ions (Hg2+). In the proposed platform, 5' thiolated 32-mer DNA probes with methylene blue at the 3' end were immobilized on a thin gold (Au) surface layer. Following Hg2+ ions interacting with T bases of the aptamer, T-Hg-T bonds are formed; resulting in a hairpin-shaped formation of the DNA and a detectable change in the IR absorbance of the sensing interface. Notably, the background noise produced by external molecules (e.g., water, non-specific binding molecules and bulk solution) is reduced to a negligible level by means of the ATR detection mode. It is shown that the proposed sensor has a linear response (R2 = 0.986) with high sensitivity and good selectivity over the Hg2+ range of 0.01 µM-50 µM.

A bead-based immunofluorescence-assay on a microfluidic dielectrophoresis platform for rapid dengue virus detection.

The proof of concept of utilizing a microfluidic dielectrophoresis (DEP) chip was conducted to rapidly detect a dengue virus (DENV) in vitro based on the fluorescence immunosensing. The mechanism of detection was that the DEP force was employed to capture the modified beads (mouse anti-flavivirus monoclonal antibody-coated beads) in the microfluidic chip and the DENV modified with fluorescence label, as the detection target, can be then captured on the modified beads by immunoreaction. The fluorescent signal was then obtained through fluorescence microscopy, and then quantified by ImageJ freeware. The platform can accelerate an immuno-reaction time, in which the on-chip detection time was 5min, and demonstrating an ability for DENV detection as low as 104 PFU/mL. Furthermore, the required volume of DENV samples dramatically reduced, from the commonly used ~50µL to ~15µL, and the chip was reusable (>50x). Overall, this platform provides a rapid detection (5min) of the DENV with a low sample volume, compared to conventional methods. This proof of concept with regard to a microfluidic dielectrophoresis chip thus shows the potential of immunofluorescence based-assay applications to meet diagnostic needs.

Real-time electrochemical recording of dopamine release under optogenetic stimulation.

Dopaminergic PC12 cells can synthesize and release dopamine, providing a good cellular model for investigating dopamine regulation. Optogenetic stimulation of channelrhodopsin-2 provides high spatial and temporal precision for selective stimulation as a powerful neuromodulation tool for neuroscience studies. The aim of this study is to measure dopamine release from dopaminergic PC12 cells under optogenetic stimulation using electrochemical recording of self-assembled monolayers modified microelectrode with amperometric measurement in real time. The activation of PC12 cells under various optogenetic stimulation schemes are characterized by measuring single-cell Ca(2+) imaging. After 10 seconds of optogenetic stimulation, the evoked intracellular Ca(2+) level and dopamine current of channelrhodopsin-2-transfected PC12 cells were 1.6- and 3.5-fold higher than those of the control cells. The optogenetic stimulation effects on Ca(2+) influx and dopamine release were 81% and 63% inhibition by using a Ca(2+) channel antagonist Nifedipine. The results indicate that optogenetic stimulation can evoke voltage-gated Ca(2+) channel-dependent dopamine exocytosis from PC12 cells in a cell specific, temporally precise and dose-dependent manner. This proposed dopamine recording system can be developed to be a good cell model for dopamine regulation and drug screening in vitro, or dopaminergic cell implantation therapy in vivo using optogenetic stimulation in a precise and convenient way.

Microelectrodes with gold nanoparticles and self-assembled monolayers for in vivo recording of striatal dopamine.

Electrochemical determination of in vivo dopamine (DA) using implantable microelectrodes is essential for monitoring the DA depletion of an animal model of Parkinson's disease (PD), but faces substantial interference from ascorbic acid (AA) in the brain area due to similar electroactive characteristics. This study utilizes gold nanoparticles (Au-NPs) and self-assembled monolayers (SAMs) to modify platinum microelectrodes for improving sensitivity and specificity to DA and alleviating AA interference. With appropriate choice of ω-mercaptoalkane carboxylic acid chain length, our results show that a platinum microelectrode coated with Au-NPs and 3-mercaptopropionic acid (MPA) has approximately an 881-fold specificity to AA. During amperometric measurements, Au-NP/MPA reveals that the responsive current is linearly dependent on DA over the range of 0.01-5 µM with a correlation coefficient of 0.99 and the sensitivity is 2.7-fold that of a conventional Nafion-coated electrode. Other important features observed include fast response time (below 2 s), resistance to albumin adhesion and low detection limit (7 nM) at a signal to noise ratio of 3. Feasibility of in vivo DA recording with the modified microelectrodes is verified by real-time monitoring of electrically stimulated DA release in the striatum of anesthetized rats with various stimulation parameters and administration of a DA uptake inhibitor. The developed microelectrodes present an attractive alternative to the traditional options for continuous electrochemical in vivo DA monitoring.