A fast and ultrasensitive ELISA based on rolling circle amplification.

A highly sensitive ELISA is critical for early diagnosis and biomarker discovery of various diseases. Although various ELISA technologies have been developed with high sensitivity, they are limited by poor repeatability, high cost, the dependence on complex equipment and/or a prolonged reaction time. To this end, we developed a fast and ultrasensitive ELISA (termed RELISA) based on rolling circle amplification (RCA) and enzymatic signal amplification. The RELISA is established on the traditional ELISA, with only one more RCA step that can be accomplished within 10 minutes. The prolonged single strand DNA (ssDNA) from RCA is able to enrich abundant horseradish peroxidase conjugate (HRP) modified detection probes. Consequently, the intensive HRP is able to catalyze TMB-H2O2 to produce significantly enhanced colorimetric signals. With CEACAM-7 as a model biomarker, the RELISA achieves the limit of detection as low as 2.82 pg mL-1, which is ∼50 times higher than that of the traditional ELISA. Therefore, we envision that the developed RELISA would be a powerful tool for the early diagnosis of various major diseases.

A digitalized isothermal nucleic acid testing platform based on a pump-free open droplet array microfluidic chip.

Digital PCR has shown great potential for quantitative nucleic acid testing (NAT), but most existing platforms are dependent on large auxiliary equipment (e.g., vacuum pump, amplification instrument, fluorescence microscope) to achieve target dispersion, amplification, signal capture and result analysis. Such complex, expensive and bulky NAT platforms have limited their applications in resource-limited areas, especially for point-of-care testing (POCT). In this work, we designed a digital isothermal NAT platform based on a pump-free open droplet array microfluidic chip. A pump-free microfluidic chip was developed based on an open microdroplet array in the form of thousands of independent microdroplets for spontaneous sample dispersion, without the need for external power. Combined with a handheld fluorescent signal reader based on a smartphone, this digital NAT platform can accurately quantify as low as 1 copy per µL of λDNA. Therefore, our integrated NAT platform, as a potable, robust and low-cost tool for highly accurate NA quantitative analysis, holds great potential for POCT applications.

Point-of-Care HIV Test for a Promising Simple and Rapid Clinical HIV Definite Diagnosis Process.

BACKGROUND: This study compared and evaluated the performance of a commercially available HIV POC rapid test with assays commonly used in clinical laboratories, including enzymelinked immunosorbent assay (ELISA), western blot (WB), and reverse transcription-polymerase chain reaction (RT-PCR). METHODS: 500 patients' samples were detected by the POC rapid test and clinically common tests (WB, ELISA, and RT-PCR) to compare detection performance, test time, and test cost. RESULTS: Taking the WB results as the gold standard, the results of RT-PCR were completely consistent with WB. The concordance of ELISA and POC with WB was 82.00% and 93.80%, respectively, with statistically significant differences (p<0.05). CONCLUSION: This study provides evidence that rapid HIV POC assays are superior to ELISA and that WB and RT-PCR have equal detection performance in detecting HIV. As a result, a rapid and costeffective HIV definition process based on the POC assays can be proposed.

Terahertz spectra of proteinuria and non-proteinuria.

In clinical practice, proteinuria detection is of great significance in the diagnosis of kidney diseases. Dipstick analysis is used in most outpatient settings to semi-quantitatively measure the urine protein concentration. However, this method has limitations for protein detection, and alkaline urine or hematuria will cause false positive results. Recently, terahertz time-domain spectroscopy (THz-TDS) with strong hydrogen bonding sensitivity has been proven to be able to distinguish different types of biological solutions, which means that protein molecules in urine may have different THz spectral characteristics. In this study, we performed a preliminary clinical study investigating the terahertz spectra of 20 fresh urine samples (non-proteinuria and proteinuria). The results showed that the concentration of urine protein was positively correlated with the absorption of THz spectra at 0.5-1.2 THz. At 1.0 THz, the pH values (6, 7, 8, and 9) had no significant effect on the THz absorption spectra of urine proteins. The terahertz absorption of proteins with a high molecular weight (albumin) was greater than that of proteins with a low molecular weight (ß2-microglobulin) at the same concentration. Overall, THz-TDS spectroscopy for the qualitative detection of proteinuria is not affected by pH and has the potential to discriminate between albumin and ß2-microglobulin in urine.

Similar color analysis based on deep learning (SCAD) for multiplex digital PCR via a single fluorescent channel.

Digital PCR (dPCR) has recently attracted great interest due to its high sensitivity and accuracy. However, the existing dPCR depends on multicolor fluorescent dyes and multiple fluorescent channels to achieve multiplex detection, resulting in increased detection cost and limited detection throughput. Here, we developed a deep learning-based similar color analysis method, namely SCAD, to achieve multiplex dPCR in a single fluorescent channel. As a demonstration, we designed a microwell chip-based diplex dPCR system for detecting two genes (blaNDM and blaVIM) with two kinds of green fluorescent probes, whose emission colors are difficult to discriminate by traditional fluorescence intensity-based methods. To verify the possibility of deep learning algorithms to distinguish the similar colors, we first applied t-distributed stochastic neighbor embedding (tSNE) to make a clustering map for the microwells with similar fluorescence. Then, we trained a Vision Transformer (ViT) model on 10 000 microwells with two similar colors and tested it with 262 202 microwells. Lastly, the trained model was proven to have highly accurate classification ability (>98% for both the training set and the test set) and precise quantification ability on both blaNDM and blaVIM (ratio difference <0.10). We envision that the developed SCAD method would significantly expand the detection throughput of dPCR without the need for other auxiliary equipment.

Artificial intelligence-assisted colorimetric lateral flow immunoassay for sensitive and quantitative detection of COVID-19 neutralizing antibody.

Currently, vaccination is the most effective medical measure to improve group immunity and prevent the rapid spread of COVID-19. Since the individual difference of vaccine effectiveness is inevitable, it is necessary to evaluate the vaccine effectiveness of every vaccinated person to ensure the appearance of herd immunity. Here, we developed an artificial intelligent (AI)-assisted colorimetric polydopamine nanoparticle (PDA)-based lateral flow immunoassay (LFIA) platform for the sensitive and accurate quantification of neutralizing antibodies produced from vaccinations. The platform integrates PDA-based LFIA and a smartphone-based reader to test the neutralizing antibodies in serum, where an AI algorithm is also developed to accurately and quantitatively analyze the results. The developed platform achieved a quantitative detection with 160 ng/mL of detection limit and 625-10000 ng/mL of detection range. Moreover, it also successfully detected totally 50 clinical serum samples, revealing a great consistency with the commercial ELISA kit. Comparing with commercial gold nanoparticle-based LFIA, our PDA-based LFIA platform showed more accurate quantification ability for the clinical serum. Therefore, we envision that the AI-assisted PDA-based LFIA platform with sensitive and accurate quantification ability is of great significance for large-scale evaluation of vaccine effectiveness and other point-of-care immunoassays.

Taqman-MGB nanoPCR for Highly Specific Detection of Single-Base Mutations.

PURPOSE: Detection of single-base mutations is important for real-time monitoring of tumor progression, therapeutic effects, and drug resistance. However, the specific detection of single-base mutations from excessive wild-type background sequences with routine PCR technology remains challenging. Our objective is to develop a simple and highly specific qPCR-based single-base mutation detection method. METHODS: Using EGRF T790M as a model, gold nanoparticles at different concentrations were separately added into the Taqman-MGB qPCR system to test specificity improvement, leading to the development of the optimal Taqman-MGB nanoPCR system. Then, these optimal conditions were used to test the range of improvement in the specificity of mutant-type and wild-type templates and the detection limit of mutation abundances in a spiked sample. RESULTS: The Taqman-MGB nanoPCR was established based on the traditional qPCR, with significantly suppressed background noise and improved specificity for single-base mutation detection. With EGFR T790M as a template, we demonstrated that our Taqman-MGB nanoPCR system could improve specificity across a wide concentration range from 10-9 µM to 10 µM and detect as low as 0.95% mutation abundance in spiked samples, which is lower than what the traditional Taqman-MGB qPCR and existing PCR methods can detect. Moreover, we also proposed an experimentally validated barrier hypothesis for the mechanism of improved specificity. CONCLUSION: The developed Taqman-MGB nanoPCR system could be a powerful tool for clinical single-base mutation detection.

Risk Factors And Clinical Outcomes Of Hospital-Acquired MRSA Infections In Chongqing, China.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen of hospital infection with multi-drug resistant characteristics. Its spread and epidemic pose great challenges to nosocomial infection control. This study was aimed to identify risk factors for hospital-acquired MRSA (HA-MRSA) infections and investigate its clinical outcome, developing infection control strategies and improving patient outcomes. METHODS: A retrospective case-case-control study was conducted to compare patients in Southwest Hospital, Chongqing, People's Republic of China from January 2018 to December 2018 with control patients. In this study, 251 patients with MRSA nosocomial infection, 339 patients with methicillin-sensitive Staphylococcus aureus strains (MSSA) nosocomial infection, and 300 patients with non-Staphylococcus aureus infection were included. RESULTS: Multivariate analysis showed that presence of central venous catheters (odds ratio [OR], 1.932; 95% confidence interval [CI], 1.074-3.477; P=0.028), sputum suction (OR, 2.887; 95% CI, 1.591-5.240; p<0.001), and total hospital stays more than 30 days (OR, 3.067; 95% CI, 2.063-4.559; P<0.001) were independent risk factors for HA-MRSA. Renal insufficiency (OR, 2.744; 95% CI, 1.089-6.914; P=0.032) and receipt of immunosuppressors (OR, 3.140; 95% CI, 1.284-7.678; P=0.012) were independent predictors of poor prognosis of MRSA nosocomial infection. Moreover, empirical use of antibiotics (OR, 0.514; 95% CI, 0.282-0.935; P=0.029) was a protective factor for poor prognosis of MRSA nosocomial infection. In-hospital mortality in the MRSA group was not statistically significant compared with the other two groups; however, the rate of poor prognosis in the MRSA group was higher than that of the MSSA group (27.5% vs 17.1%, χ 2=9.200, P=0.002) and the control group (27.5% vs 16.0%, χ 2=19.190, P=0.001). CONCLUSION: Our results have shown presence of central venous catheters, sputum suction, and total hospital stays more than 30 days were associated with nosocomial MRSA infection. Patients with renal insufficiency and immunosuppressive therapy were more likely to cause poor prognosis with MRSA infection, and the empirical use of antibiotics can effectively reduce the adverse clinical outcomes caused by MRSA infection. Based on above findings, strategies to control MRSA infection should emphasize more attention to these patients and appropriate empirical use of antibiotics.