Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification.

Using rolling circle amplification (RCA) and two different ways of signal readout, we developed analytical methods to detect the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S protein). We modified streptavidin-coated magnetic beads with an aptamer of RBD through a biotin-tagged complementary DNA strand (biotin-cDNA). Binding of RBD caused the aptamer to dissociate from the biotin-cDNA, making the cDNA available to initiate RCA on the magnetic beads. Detection of RBD was achieved using a dual signal output. For fluorescence signaling, the RCA products were mixed with a dsDNA probe labeled with fluorophore and quencher. Hybridization of the RCA products caused the dsDNA to separate and to emit fluorescence (λex = 488 nm, λem = 520 nm). To generate easily detectable UV-vis absorbance signal, the RCA amplification was extended to produce DNA flower to encapsulate horseradish peroxidase (HRP). The HRP-encapsulated DNA flower catalyzed a colorimetric reaction between H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) to generate an optical signal (λabs = 450 nm). The fluorescence and colorimetric assays for RBD have low detection limits (0.11 pg mL-1 and 0.904 pg mL-1) and a wide linear range (0.001-100 ng mL-1). For detection of RBD in human saliva, the recovery was 93.0-100% for the fluorescence assay and 87.2-107% for the colorimetric assay. By combining fluorescence and colorimetric detection with RCA, detection of the target RBD in human saliva was achieved with high sensitivity and selectivity.

COVID-19 Detection Using a 3D-Printed Micropipette Tip and a Smartphone.

The COVID-19 pandemic has caused over 7 million deaths worldwide and over 1 million deaths in the US as of October 15, 2022. Virus testing lags behind the level or availability necessary for pandemic events like COVID-19, especially in resource-limited settings. Here, we report a low cost, mix-and-read COVID-19 assay using a synthetic SARS-CoV-2 sensor, imaged and processed using a smartphone. The assay was optimized for saliva and employs 3D-printed micropipette tips with a layer of monoclonal anti-SARS-CoV-2 inside the tip. A polymeric sensor for SARS-CoV-2 spike (S) protein (COVRs) synthesized as a thin film on silica nanoparticles provides 3,3',5-5'-tetramethylbenzidine responsive color detection using streptavidin-poly-horseradish peroxidase (ST-poly-HRP) with 400 HRP labels per molecule. COVRs were engineered with an NHS-PEG4-biotin coating to reduce nonspecific binding and provide affinity for ST-poly-HRP labels. COVRs binds to S-proteins with binding strengths and capacities much larger than salivary proteins in 10% artificial saliva-0.01%-Triton X-100 (as virus deactivator). A limit of detection (LOD) of 200 TCID50/mL (TCID50 = tissue culture infectious dose 50%) in artificial saliva was obtained using the Color Grab smartphone app and verified using ImageJ. Viral load values obtained in 10% pooled human saliva spiked with inactivated SARS-COV-2 virus gave excellent correlation with viral loads obtained from qPCR (p = 0.0003, r = 0.99).

Portable high-throughput multimodal immunoassay platform for rapid on-site COVID-19 diagnostics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a causal agent of Coronavirus Disease 2019 (COVID-19) has led to the global pandemic. Though the real-time reverse transcription polymerase chain reaction (RT-PCR) acting as a gold-standard method has been widely used for COVID-19 diagnostics, it can hardly support rapid on-site applications or monitor the stage of disease development as well as to identify the infection and immune status of rehabilitation patients. To suit rapid on-site COVID-19 diagnostics under various application scenarios with an all-in-one device and simple detection reagents, we propose a high-throughput multimodal immunoassay platform with fluorescent, colorimetric, and chemiluminescent immunoassays on the same portable device and a multimodal reporter probe using quantum dot (QD) microspheres modified with horseradish peroxidase (HRP) coupled with goat anti-human IgG. The recombinant nucleocapsid protein fixed on a 96-well plate works as the capture probe. In the condition with the target under detection, both reporter and capture probes can be bound by such target. When illuminated by excitation light, fluorescence signals from QD microspheres can be collected for target quantification often at a fast speed. Additionally, when pursuing simple detection without using any sensing devices, HRP-catalyzed TMB colorimetric immunoassay is employed; and when pursuing highly sensitive detection, HRP-catalyzed luminol chemiluminescent immunoassay is established. Verified by the anti-SARS-CoV-2 N humanized antibody, the sensitivities of colorimetric, fluorescent, and chemiluminescent immunoassays are respectively 20, 80, and 640 times more sensitive than that of the lateral flow colloidal gold immunoassay strip. Additionally, such a platform can simultaneously detect multiple samples at the same time thus supporting high-throughput sensing; and all these detecting operations can be implemented on-site within 50 min relying on field-operable processing and field-portable devices. Such a high-throughput multimodal immunoassay platform can provide a new all-in-one solution for rapid on-site diagnostics of COVID-19 for different detecting purposes.

Enhancing CRISPR-Cas-Mediated Detection of Nucleic Acid and Non-nucleic Acid Targets Using Enzyme-Labeled Reporters.

We introduce a new method to generate an amplified signal in CRISPR-Cas-based detection. Target recognition activates a CRISPR-Cas complex, leading to catalytic cleavage of horseradish peroxidase (HRP)-labeled oligonucleotides from the surface of microbeads. We show that the HRP released into solution can be monitored through colorimetric, fluorometric, or luminescent approaches, yielding up to ∼75-fold turn-on signal and limits of detection (LODs) as low as ∼10 fM. Compared to Cas-based detection with a conventional fluorophore/quencher reporter, this strategy improves the LOD by ∼30-fold. As a proof-of-concept, we show the rapid (<1 h), PCR-free, and room temperature (25 °C) detection of a nucleic acid marker for the SARS-CoV-2 virus with the naked eye at clinically relevant concentrations. We further show that the probe set can be programmed to be recognized and activated in the presence of non-nucleic acid targets. Specifically, we show adenosine triphosphate (ATP) binding to an aptamer can activate CRISPR-Cas and trigger a colorimetric readout, enabling the analysis of ATP in human serum samples with sensitivity on par with that of several commercially available kits. Taken together, the strategy reported herein offers a simple and sensitive platform to detect analytes where target amplification is either inconvenient (e.g., PCR under point-of-care settings) or impossible.

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum.

Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55-1.65% and 2.58-6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum.

Direct in situ fluorescent enzyme-linked immunosorbent assay (ELISA) is rarely investigated and reported. Herein, a direct in situ high-performance HRP-labeled fluorescent immunoassay platform was constructed. The platform was developed based on a rapid in situ fluorogenic reaction between Polyethyleneimine (PEI) and p-Phenylenediamine (PPD) analogues to generate fluorescent copolymer nanoparticles (FCNPs). The formation mechanism of FCNPs was found to be the oxidation of •OH radicals, which was further proved by nitrogen protection and scavenger of •OH radicals. Meantime, the fluorescence wavelength of FCNPs could be adjusted from 471 to 512 nm by introducing various substitution groups into the PPD structure. Using cardiac troponin I (cTnI) and SARS-CoV-2 nucleocapsid protein (N-protein) as the model antigens, the proposed fluorescent ELISA exhibited a wide dynamic range of 5-180 ng/mL and a low limit of detection (LOD) of 0.19 ng/mL for cTnI, and dynamic range of 0-120 ng/mL and a LOD of 0.33 ng/mL for SARS-CoV-2 N protein, respectively. Noteworthy, the proposed method was successful applied to evaluate the cTnI and SARS-CoV-2 N protein levels in serum with satisfied results. Therefore, the proposed platform paved ways for developing novel fluorescence-based HRP-labeled ELISA technologies and broadening biomarker related clinical diagnostics.

Synthesis of pH and Glucose Responsive Silk Fibroin Hydrogels.

Silk fibroin (SF) has attracted much attention due to its high, tunable mechanical strength and excellent biocompatibility. Imparting the ability to respond to external stimuli can further enhance its scope of application. In order to imbue stimuli-responsive behavior in silk fibroin, we propose a new conjugated material, namely cationic SF (CSF) obtained by chemical modification of silk fibroin with ε-Poly-(L-lysine) (ε-PLL). This pH-responsive CSF hydrogel was prepared by enzymatic crosslinking using horseradish peroxidase and H2O2. Zeta potential measurements and SDS-PAGE gel electrophoresis show successful synthesis, with an increase in isoelectric point from 4.1 to 8.6. Fourier transform infrared (FTIR) and X-ray diffraction (XRD) results show that the modification does not affect the crystalline structure of SF. Most importantly, the synthesized CSF hydrogel has an excellent pH response. At 10 wt.% ε-PLL, a significant change in swelling with pH is observed. We further demonstrate that the hydrogel can be glucose-responsive by the addition of glucose oxidase (GOx). At high glucose concentration (400 mg/dL), the swelling of CSF/GOx hydrogel is as high as 345 ± 16%, while swelling in 200 mg/dL, 100 mg/dL and 0 mg/dL glucose solutions is 237 ± 12%, 163 ± 12% and 98 ± 15%, respectively. This shows the responsive swelling of CSF/GOx hydrogels to glucose, thus providing sufficient conditions for rapid drug release. Together with the versatility and biological properties of fibroin, such stimuli-responsive silk hydrogels have great potential in intelligent drug delivery, as soft matter substrates for enzymatic reactions and in other biomedical applications.

Reversible Lectin Binding to Glycan-Functionalized Graphene.

The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands-chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate-lectin binding.

Detection of the SARS-CoV-2 humanized antibody with paper-based ELISA.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng µL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.