Ultrasensitive and point-of-care detection of plasma phosphorylated tau in Alzheimer’s disease using colorimetric and surface-enhanced Raman scattering dual-readout lateral flow assay

Phosphorylation of tau at Ser(396,404)(p-tau^(396,404))is one of the earliest phosphorylation events,plasma p-tau^(396,404) level appears to be a potentially promising biomarker of Alzheimer’s disease(AD).The low abundance and easy degradation of p-tau in the plasma make the lateral flow assay(LFA)a suitable choice for point-of-care detection of plasma p-tau^(396,404) levels.Herein,based on our screening of a pair of p-tau^(396,404)-specific antibodies,we developed a colorimetric and surface-enhanced Raman scattering(SERS)dual-readout LFA for the rapid,highly sensitive,robust detection of plasma p-tau^(396,404) levels.This LFA realized a detection limit of 60 pg/mL by the naked eye or 3.8 pg/mL by SERS without cross-reacting with other tau species.More importantly,LFA rapidly and accurately differentiated AD patients from healthy controls,suggesting that it has the potential for clinical point-of-care application in AD diagnosis.This dual-readout LFA has the advantages of simple operation,rapid,ultra-sensitive detection,providing a new way for early AD diagnosis and intervention,especially in primary and community AD screening.

State of the art:Lateral flow assay(LFA) biosensor for on-site rapid detection

Recently, lateral flow assay （LFA） has attracted researchers＇ attention because of its considerable advantages of superior portability, rapid detection, cost-effectiveness and ease of use. This review provides a brief overview of latest researches of LFA, including the practical use of LFA in both qualitative and quantitative analysis in different areas. Though bio-recognition molecules in the LFA used to be antibodies, a new kind of recognition element called aptamer, showing significant advantages, is developed rapidly in recent years. The highly specific recognition of aptamers/antibodies and targets are combined with the excellent properties of a dry-reagent strip biosensor that enables efficiently detection in point-of-care applications. Herein, we compared the aptamers with antibodies, summarized the principle of LFAs, and three main elements for the LFAs （recognition molecule, signal transduction element, the targets）. Additionally, we summarized different optimal experimental conditions in the recent LFA-related studies to give detailed overview of the LFA development. We hope the review can give a general guide for the development of LFAs.

A new quality control method for lateral flow assay

Precision and repeatability are challenging issues in point of care testing(POCT) analysis. Herein, we proposed a lateral flow assay(LFA) based on internal quality control microspheres to realize the accurate diagnosis of HbAlc in human body. Fluorescein cy5 decorated microspheres are used as labels for HbAlc detection, and BSA-fluorescein isothiocyanate(FITC) decorated microspheres are used as internal quality control labels. One test line was employed in the strip for the detection of glycosylated hemoglobin(HbAlc). This method can eliminate the interference of environmental factors(temperature, humidity,etc.) to LFA in the process of chromatography, and improve the precision and accuracy of HbAlc detection.The CV for detection of low concentration HbAlc was 1.05%, and the CV for detection of high concentration HbAlc was 0.69%. We envision the method to have great prospect in in vitro diagnosis(IVD).

A Novel Molecular Imprinted Polymers-Based Lateral Flow Strip for Sensitive Detection of Thiodiglycol

Traditional detection of thiodiglycol(TDG),a metabolic marker for sulfur mustard poisoning,requires not only professional operators,but also expensive reagents and large instruments.Herein,we developed a novel molecular imprinted polymers(MIPs)-based lateral flow assay(LFA)strategy for the quick,sensitive,and selective detection of TDG.Gold nanoparticles(Au NPs),MIPs,and metallothioneins(MTs)were respectively loaded on the conjugate pad,test line(T line)and control line(C line).After adding TDG,Au NPs on the conjugate pad reacted with TDG through the Au-S bond first.Then,under the action of capillary force,the conjugates of TDG and Au NPs were trapped by the MIPs as they traveled to the T line,and the residual Au NPs bound with the MTs on the C line,exhibiting two obvious red bands on T line and C line,respectively.In contrast,a single red band could be observed on C line without TDG.This method exhibited a wide linear range from 10.0 pg/m L to 10,000.0 ng/m L and its limit of detection(LOD)was as low as 0.41 pg/m L.This method was successfully utilized to detect TDG in human urine,presenting significant potential in the point-of-care testing of TDG in clinical samples of the sulfur mustard poisoning patients.

A New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope

Objective To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic （POCT） device with wide scope. Methods The lateral flow assay （LFA） strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit. Results Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit. Conclusion The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment.

Super-assembly of integrated gold magnetic assay with loopmediated isothermal amplification for point-of-care testing

With the increasing global threat of various diseases and infections,it is essential to develop a fast,low-cost,and easy-to-use point-of-care testing(POCT)system for inspections at all levels of medical institutions and self-examination at home.In this work,gold magnetic nanoparticles(GMNPs)are used as the key material,and a rapid visual detection method is designed through integrating loop-mediated isothermal amplification(LAMP)and lateral flow assay(LFA)biosensor for detecting a variety of analytes which includes whole blood,buccal swabs,and DNA.It is worth to note that the proposed method does not need DNA extraction.Furthermore,uracil DNA glycosylase(UDG)is employed to eliminate carrier contamination for preventing false positive results.The whole detection process can be finished within 25 min.The accuracy of detection is measured by assessing the polymorphisms of the methylenetetrahydrofolate reductase(MTHFR)C677T.The detection limit of the newly developed extraction-free detection system for MTHFR C677T is 0.16 ng/μL.A preliminary clinical study of the proposed method is carried out by analyzing 600 clinical samples(including 200 whole blood samples,100 buccal swabs,and 300 genomic DNA samples).The results indicate that the proposed method is 100%consistent with the sequencing results which provides a new choice for POCT and shows a broad application prospect in all levels of medical clinics and at home.

Sensitively detecting antigen of SARS-CoV-2 by NIR-Ⅱ fluorescent nanoparticles

Early detection of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection is an efficient way to prevent the spread of coronavirus disease 2019(COVID-19).Detecting SARS-CoV-2 antigen can be rapid and convenient,but it is still challenging to develop highly sensitive methods for effective diagnosis.Herein,a lateral flow assay(LFA)based on fluorescent nanoparticles emitting in the second near-infrared(NIR-II)window is developed for sensitive detection of SARS-CoV-2 antigen.Benefiting from the NIR-II fluorescence with high penetration and low autofluorescence,such NIR-II based LFA allows enhanced signal-to-background ratio,and the limit of detection is down to 0.01 ng·mL^(−1)of SARS-CoV-2 antigen.In the clinical swab sample tests,the NIR-II LFA outperforms the colloidal gold LFA with higher overall percent agreement with the polymerase chain reaction test.The clinical samples with low antigen concentrations(~0.015–~0.068 ng·mL^(−1))can be successfully detected by the NIR-II LFA,but fail for the colloidal gold LFA.The NIR-II LFA can provide a promising platform for highly sensitive,rapid,and cost-effective method for early diagnosis and mass screening of SARS-CoV-2 infection.