Application of the amplification-free SERS-based CRISPR/Cas12a platform in the identification of SARS-CoV-2 from clinical samples.

The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).

A filter pad design-based multiplexed lateral flow immunoassay for rapid simultaneous detection of PDCoV, TGEV, and PEDV in swine feces.

Swine Enteric Coronaviruses (SECoVs), with high lethality and infectiousness, are the main pathogens causing fatal and watery diarrhea in piglets and spreading globally. Moreover, these SECoVs can cause similar clinical manifestations and are often co-infected, requiring an accurate assay suitable for rapid, in situ, and differential detection. Here, we developed a multiplexed fluorescent-based lateral flow immunoassay (mFB-LFIA) for the detection of three SECoVs, including porcine delta coronaviruses (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV), in swine fecal samples. Thanks to the filter pad design and reasonable optimization, the mFB-LFIA was achieved within 15 min for three SECoVs detection simultaneously and improved the tolerance of the strips for feces samples. The limit of detection (LoD) of detecting PDCoV, TGEV, and PEDV were 2.1 × 104 TCID50 mL-1, 3.4 × 102 TCID50 mL-1, and 3.6 × 102 TCID50 mL-1, respectively. Additionally, the proposed assay was successfully applied to the detection of PDCoV, TGEV, and PEDV in swine feces with high accuracy. Compared with the gold standard nucleic acid testing, the total coincidence rate of the proposed assay was more than 90 %. Moreover, the mFB-LFIA performed excellent stability and repeatability. The proposed mFB-LFIA allows for rapid, in situ, more cost-effective and simultaneous detection of PDCoV, TGEV, and PEDV compared with nucleic acid testing. To the best of our knowledge, this is the first report to describe a multiplexed point-of-care assay capable of detecting PDCoV, TGEV, and PEDV in swine fecal samples. We believe our approach has a great potential for application to pig farm.

Rapid Evaluation of Lung Adenocarcinoma Progression by Detecting Plasma Extracellular Vesicles with Lateral Flow Immunoassays.

Extracellular vesicles (EVs) have been widely used in liquid biopsy to diagnose and monitor cancers. However, since samples containing EVs are usually body fluids with complex components, the cumbersome separation steps for EVs during detection limit the clinical application and promotion of EV detection methods. In this study, a dyad lateral flow immunoassay (LFIA) strip for EV detection, containing CD9-CD81 and EpCAM-CD81, was developed to detect universal EVs and tumor-derived EVs, respectively. The dyad LFIA strip can directly detect trace plasma samples and effectively distinguish the cancerous sample from healthy plasma. The limit of detection for detecting universal EVs was 2.4 × 105 mL-1. The whole immunoassay can be performed in 15 min and only consumes 0.2 µL of plasma for one test. To improve the suitability of a dyad LFIA strip in complex scenarios, a smartphone-based photographic method was developed, which provided a consistency of 96.07% to a specialized fluorescence LFIA strip analyzer. In further clinical testing, EV-LFIA discriminated lung cancer patient groups (n = 25) from healthy controls (n = 22) with 100% sensitivity and 94.74% specificity at the best cutoff. The detection of EpCAM-CD81 tumor EVs (TEVs) in lung cancer plasma revealed the differences in TEVs in individuals, which reflected the different treatment effects. TEV-LFIA results were compared with CT scan findings (n = 30). The vast majority of patients with increased TEV-LFIA detection intensity had lung masses that enlarged or remained unchanged in size, which reported no response to treatment. In other words, patients who reported no response (n = 22) had a high TEV level compared with patients who reported a response to treatment (n = 8). Taken together, the developed dyad LFIA strip provides a simple and rapid platform to characterize EVs to monitor lung cancer therapy outcomes.

Platinum nanoparticles (PtNPs)-based CRISPR/Cas12a platform for detection of nucleic acid and protein in clinical samples.

Early rapid screening diagnostic assay is essential for the identification, prevention, and evaluation of many contagious or refractory diseases. The optical density transducer created by platinum nanoparticles (PtNPs) (OD-CRISPR) is reported in the present research as a cheap and easy-to-execute CRISPR/Cas12a-based diagnostic platform. The OD-CRISPR uses PtNPs, with ultra-high peroxidase-mimicking activity, to increase the detection sensitivity, thereby enabling the reduction of detection time and cost. The OD-CRISPR can be utilized to identify nucleic acid or protein biomarkers within an incubation time of 30-40min in clinical specimens. In the case of taking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene as an instance, when compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR), the OD-CRISPR test attains a sensitivity of 79.17% and a specificity of 100%. In terms of detecting prostate-specific antigen (PSA), aptamer-based OD-CRISPR assay achieves the least discoverable concentration of 0.01 ng mL-1. In general, the OD-CRISPR can detect nucleic acid and protein biomarkers, and is a potential strategy for early rapid screening diagnostic tools.

CD8 cell counting in whole blood by a paper-based time-resolved fluorescence lateral flow immunoassay.

The number of CD8+ T lymphocytes (CD8 cells) in peripheral blood can directly reflect the immune status of the body and is widely used for auxiliary diagnosis and prognostic evaluation of diseases. There is an urgent need to develop a simple CD8 cell-counting platform to meet clinical needs. Our group designed a paper-based cell-counting method based on a blocking competition strategy. In addition, we developed a time-resolved fluorescence-blocking competitive lateral flow immunoassay (TRF-BCLFIA) for point-of-care CD8 cell counting that functions by measuring europium nanoparticle (EuNP)-labeled CD8 antibody probes that are not captured by CD8 cells, and we indirectly calculated the concentration of CD8 cells in samples. Within 30 min, four operation steps can provide an accurate CD8 cell count for a 75-µL whole-blood sample, and this approach can be implemented on a handheld device. The TRF-BCLFIA reliably quantified CD8 cells in whole-blood samples, in which the assay exhibited a linear correlation (R2 = 0.989) readout for CD8 cell concentrations ranging from 137 to 821 cells/µL. To validate this approach, our newly developed CD8 cell-counting tool was used to assess 33 tumor patient blood samples. The results showed a high consistency with a flow cytometry-based absolute count. This analysis approach is a promising alternative for the costly standard flow cytometry-based tools for CD8 cell counting in tumor patients in community clinics, small hospitals, and low medical resource regions. This technology would deliver simple diagnostics to patients anywhere in the world, regardless of geography or socioeconomic status.