A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay.

The digital immunoassay is a highly sensitive detection technique based on single-molecule counting and is widely used in the ultrasensitive detection of biomarkers. Herein, we developed a fluorescent microsphere-based digital immunoassay (FMDIA) by employing fluorescent microspheres as both the carriers for immunoreaction and fluorescent reports for imaging. In this approach, the target protein in the sample was captured by fluorescent microspheres to form a biotin-labeled sandwich immunocomplex, and then, the fluorescent microspheres containing the target protein molecules were captured by adding streptavidin-coated magnetic beads (SA-MBs). By counting the proportion of fluorescence-positive magnetic beads, the concentration of the target protein can be precisely quantified. As a proof of concept, α fetoprotein (AFP) and human interleukin-6 (IL-6) were used to assess the analytical performance of the proposed FMDIA, and limit of detection (LOD) values of 21 pg/mL (0.30 pM) and 0.19 pg/mL (7.3 fM) were achieved, respectively. The results of AFP detection in serum samples of patients and healthy people were consistent with the reference values given by the hospital. Furthermore, by adding fluorescent microspheres of various colors for encoding, the proposed FMDIA can easily realize the simultaneous detection of multiple proteins without the need to introduce multiple modified magnetic beads. This multiplex protein detection strategy, in which the reactions are first carried out on the fluorescent microspheres and then magnetic beads are used to capture the fluorescent reporters containing the target molecules, provides a new idea for digital assays.

Amplification-free detection of Mpox virus DNA using Cas12a and multiple crRNAs.

Mpox virus (MPXV) is a zoonotic DNA virus that caused human Mpox, leading to the 2022 global outbreak. MPXV infections can cause a number of clinical syndromes, which increases public health threats. Therefore, it is necessary to develop an effective and reliable method for infection prevention and control of epidemic. Here, a Cas12a-based direct detection assay for MPXV DNA is established without the need for amplification. By targeting the envelope protein gene (B6R) of MPXV, four CRISPR RNAs (crRNAs) are designed. When MPXV DNA is introduced, every Cas12a/crRNA complex can target a different site of the same MPXV gene. Concomitantly, the trans-cleavage activity of Cas12a is triggered to cleave the DNA reporter probes, releasing a fluorescence signal. Due to the application of multiple crRNAs, the amount of active Cas12a increases. Thus, more DNA reporter probes are cleaved. As a consequence, the detection signals are accumulated, which improves the limit of detection (LOD) and the detection speed. The LOD of the multiple crRNA system can be improved to ~ 0.16 pM, which is a decrease of the LOD by approximately ~ 27 times compared with the individual crRNA reactions. Furthermore, using multiple crRNAs increases the specificity of the assay. Given the outstanding performance, this assay has great potential for Mpox diagnosis.

Nucleic Acid Amplification-Free Digital Detection Method for SARS-CoV-2 RNA Based on Droplet Microfluidics and CRISPR-Cas13a.

Most of the methods currently developed for RNA detection based on CRISPR were combined with nucleic acid amplification. As a result, such methods inevitably led to certain disadvantages such as multiple operations, expensive reagents, and amplification bias. To solve the above problems, we developed a highly sensitive and specific nucleic acid amplification-free digital detection method for SARS-CoV-2 RNA based on droplet microfluidics and CRISPR-Cas13a. In this assay, thousands of monodisperse droplets with a size of 30 µm were generated within 2 min by a negative pressure-driven microfluidic chip. By confining a single target RNA recognition event to an independent droplet, the collateral cleavage products of activated Cas13a could be accumulated in one droplet. By combining the droplet microfluidics and CRISPR-Cas13a, SARS-CoV-2 RNA could be easily detected within 30 min with a detection limit of 470 aM. The performance of this assay was verified by specificity experiments and spiking and recovery experiments with human saliva. Compared with many developed methods for SARS-CoV-2 RNA detection, our method is time- and reagent-saving and easy to operate. Taken together, this digital detection method based on droplet microfluidics and CRISPR-Cas13a provides a promising approach for RNA detection in clinical diagnostics.

CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection.

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARS-CoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

A functional nanoflower based lateral flow immunoassay for the rapid and robust detection of pathogens.

In the face of complex public health emergencies and various social medical needs in new situations, it is urgent to establish rapid detection technology for the early detection of pathogens to control their spread and minimize the resultant health and societal impact. Point-of-care testing (POCT) that allows rapid, on-site, and affordable detection and monitoring of health conditions at home or away from clinical labs has received increasing attention in modern medicine. In this work, we have synthesized multifunctional magainin I-human chorionic gonadotropin (hCG)-Cu3(PO4)2 nanoflowers and demonstrated a new strategy for the fast diagnosis of pathogenic microorganisms by combining functional nanoflowers with a lateral flow immunoassay device. The prepared multifunctional nanoflowers immobilized many signal molecules, which solves the poor sensitivity of traditional lateral flow strips and realizes the highly sensitive detection of pathogenic microorganisms ("accurate detection"). Besides, this method can complete the rapid transformation of commercial-off-the-shelf lateral flow strips and realize the fast diagnosis of target analytes in case of an outbreak ("fast detection"). Therefore, the established rapid and highly sensitive lateral flow immunoassay for the detection of pathogenic microorganisms will effectively improve the early diagnosis efficiency of infectious diseases caused by pathogenic microorganisms and shorten the diagnosis time of diseases.

Novel Method of Clickable Quantum Dot Construction for Bioorthogonal Labeling.

Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.

DNAzyme Walker for Homogeneous Detection of Enterovirus EV71 and CVB3.

When dealing with infectious pathogens, the risk of contamination or infection in the process of detecting them is nonnegligible. Separation-free detection will be beneficial in operation and safety. In this work, we proposed a DNAzyme walker for homogeneous and isothermal detection of enterovirus. The DNAzyme is divided into two inactivate subunits. When the subunit-conjugated antibody binds to the target virus, the activity of the DNAzyme recovers as a result of spatial proximity. The walker propels, and the fluorescence recovers. The final fluorescence intensity of the reaction mixture is related to the concentration of the target virus. The detection limit of this proposed method is 6.6 × 104 copies/mL for EV71 and 4.3 × 104 copies/mL for CVB3, respectively. Besides, this method was applied in detection of EV71 in clinical samples with a satisfactory result. The entire experiment is easy to operate, and the proposed method has great potential for practical use.

A fluorescence color card for point-of-care testing (POCT) and its application in simultaneous detection.

Diabetes mellitus has received much attention because its complications include liver, kidney, eye, heart and cerebrovascular diseases. Thus, it would be highly significant to develop a rapid and efficient method for glucose detection in biological samples. In this work, a point-of-care testing (POCT) method of glucose detection was proposed using a standard colorimetric card for semi-quantitative determination patterns. In the prepared fluorescence color card for glucose, a good linear relationship was acquired by plotting the ratio of the grayscale value (I/I0) versus the logarithm of glucose concentration within 100.0 to 1000.0 µmol L-1, and the LOD of glucose detection was 1.1 µmol L-1. A large number of actual samples (30 serum and 7 urine) were analyzed and the results demonstrated that this method had good potential to be applied in the primary screening of diabetic patients. In addition, this method is universal and can be applied in the simultaneous detection of multiple small molecules. It provides a new strategy for the primary screening of multiple diseases simultaneously, which presents excellent application potential.

Point-of-care testing (POCT) of patients with a high concentration of uric acid by using alginate hydrogel microspheres embedded with CdZnTeS QDs and urate oxidase (Alg@QDs-UOx MSs).

High concentration of uric acid is usually related to cardiovascular and cerebrovascular diseases. Developing a simple method for the rapid and efficient detection of uric acid has a great significance in clinical diagnosis. In this work, alginate hydrogel microspheres embedded with CdZnTeS QDs and urate oxidase (Alg@QDs-UOx MSs) were prepared for the first time, and further used for point-of-care testing (POCT) of patients with a high concentration of uric acid. This strategy is mainly based on visual detection of H2O2, the product of uric acid after an enzymatic reaction. The proposed sensor (Alg@QDs-UOx MSs) has several advantages. First, it can reduce the interference of the proteins to the fluorescence of QDs. Second, Alg@QDs-UOx MSs help improve the stability of the CdZnTeS QDs as well as the activity of urate oxidase during storage. Third, it is easy to use, has fast response speed, and is of low cost. Therefore, the proposed sensor shows good application prospects. Simply through the built-in camera of a smartphone, we can visualize the urine samples from patients with a high concentration of uric acid within 10 minutes, and the accuracy rates were 100%. In the range of 100.0 µM to 900.0 µM, the I/I0 values and uric acid concentrations are in a great linear relationship (R2 = 0.9973), indicating that this method can be employed for quantitative analysis of uric acid in human urine (<10 mM). The limit of detection (LOD) is 20.3 µM.

Glow-type chemiluminescent hydrogels for point-of-care testing (POCT) of cholesterol.

Cholesterol is an essential compound for human health, and a high or low concentration of cholesterol is closely related to various diseases. Thus, developing a simple method for POCT of cholesterol has great significance in clinical diagnosis. In this work, alginate (Alg) hydrogels with glow-type chemiluminescence (CL) were prepared and applied for rapid and quantitative cholesterol detection via a smartphone. The glow-type CL hydrogels (HRP/COD/luminol/Alg hydrogels) contained luminol as a chemiluminescent reagent, horseradish peroxidase (HRP) and cholesterol oxidase (COD) for enzymatic cascade reactions. The HRP/COD/luminol/Alg hydrogels exhibited outstanding stability, which effectively avoided the enzyme inactivation during long-term storage. Furthermore, the HRP/COD/luminol/Alg hydrogels exhibited longer and more stable glow-type CL. With the help of COD catalytic specificity for cholesterol and bi-enzymatic cascade reactions, the glow-type CL hydrogels realized the specific and sensitive detection of cholesterol. The smartphone was used as a detector instead of a special large instrument for responding to the glow-type CL emission, and a LOD of 7.2 µM was obtained. Therefore, the proposed sensor expands the application of the glow-type CL in POCT and provides an alternative way for cholesterol detection in clinical diagnosis.

IFITM3/STAT3 axis promotes glioma cells invasion and is modulated by TGF-ß.

Glioma is the most aggressive primary brain tumor. We have previously provided evidence that IFITM3 promoted glioma cells migration. However, the mechanism of how IFITM3 regulates glioma cells invasion and whether IFITM3 participates in TGF-ß-mediated glioma invasion are still unknown. In this paper, we proved that IFITM3 was notably up-regulated in glioma tissues. Knockdown of IFITM3 suppressed STAT3 phosphorylation in vitro, and a specific STAT3 inhibitor AG490 reversed IFITM3-induced invasion of glioma cells. Furthermore, IFITM3 expression was induced by TGF-ß in glioma and IFITM3 knockdown abolished TGF-ß-mediated glioma cells invasion. Collectively, the results indicate that IFITM3/STAT3 axis may promote TGF-ß-induced glioma cells invasion. This study provided some suggestions for the clinical treatment of the brain tumor.

Homogeneous immunoassay for alpha-fetoprotein based on the quenching of the fluorescence of quantum dots by antibody labelled with complexed copper ion tags.

A homogeneous fluorescent immunoassay is described for the determination of alpha fetoprotein (AFP) relying on the interaction between copper ion complex and quantum dots (QDs). The copper ion complex-labelled antibody can be employed as a quencher of fluorescence of QDs and capture probe of AFP in homogeneous solution. The labelled antibody is mixed with QDs to form the immune ensemble probe. Upon the addition of AFP, the labelled antibody is stripped away from QDs by antigen-antibody combination leading to the increase in the fluorescence signal. Thus, the determination of AFP can be realized by fluorometry (best measured at excitation/emission wavelengths of 360/520 nm). The fluorescence intensity shows a good linear relationship with the AFP concentration ranging from 40 to 640 ng mL-1, and the LOD is 26 ng mL-1. The proposed method provides a new approach to incorporate metal complexes into QD-based biomolecule sensing. Graphical abstract Schematic presentation of a fluorescent probe comprised of quantum dots and antibody labelled with copper ion complex for homogeneous immunoassay of α-fetoprotein. The target antigen can break up the ground state QD/labelled antibody complex to set free the fluorescent QDs.

LncRNA-ATB promotes TGF-ß-induced glioma cells invasion through NF-κB and P38/MAPK pathway.

Glioma constitutes the most aggressive primary intracranial malignancy in adults. We previously showed that long noncoding RNA activated by TGF-ß (lncRNA-ATB) promoted the glioma cells invasion. However, whether lncRNA-ATB is involved in TGF-ß-mediated invasion of glioma cells remains unknown. In this study, quantitative real-time polymerase chain reaction and western blot analysis were used for detecting the mRNA and protein expression of related genes, respectively. Transwell assay was performed to assess the impact of lncRNA-ATB on TGF-ß-induced glioma cells migration and invasion. Immunofluorescence staining was utilized to characterize related protein distribution. Results showed that TGF-ß upregulated lncRNA-ATB expression in glioma LN-18 and U251 cells. Overexpression of lncRNA-ATB activated nuclear factor-κB (NF-κB) pathway and promoted P65 translocation into the nucleus, thus facilitated glioma cells invasion stimulated by TGF-ß. Similarly, lncRNA-ATB markedly enhanced TGF-ß-mediated invasion of glioma cells through activation P38 mitogen-activated protein kinase (P38/MAPK) pathway. Moreover, both the NF-κB selected inhibitor pyrrolidinedithiocarbamate ammonium and P38/MAPK specific inhibitor SB203580 partly reversed lncRNA-ATB induced glioma cells invasion mediated by TGF-ß. Collectively, this study revealed that lncRNA-ATB promotes TGF-ß-induced glioma cell invasion through NF-κB and P38/MAPK pathway and established a detailed framework for understanding the way how lncRNA-ATB performs its function in TGF-ß-mediated glioma invasion.

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging.

Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

Target-Induced Cascade Amplification for Homogeneous Virus Detection.

Detection of viruses with high sensitivity is critical for the prevention and treatment of the related disease. Two homogeneous target-induced cascade amplification methods were proposed for the detection of enterovirus 71 and coxsackievirus B3. These methods both employ DNAzyme but differ in the way in which the DNAzyme is amplified. In the hybridization chain reaction (HCR)-based strategy, the DNAzyme is assembled by hairpin DNA strands, while in the rolling circle amplification (RCA)-based strategy, the DNAzyme is synthesized by the polymerase. On the basis of the virion structure, we investigated the effects of using only VP1-antibody or VP1-antibody and VP2-antibody on the detection. And the combination of two kinds of antibodies was found to further improve the performance of the detection. Subsequently, the simultaneous detection of EV71 and CVB3 was achieved by the RCA-based strategy. And the proposed methods were also applied in clinical samples analysis with a satisfactory result, showing great potential for applications in virus detection.

The behavior of a bipedal DNA walker moving on the surface of magnet microparticles and its application in DNA detection.

In this work, a three-dimensional DNA machine based on the isothermal strand-displacement polymerase reaction (ISDPR) has been constructed. The walking behavior of a DNA walker on the obstructive surface of magnetic beads has also been studied by adding different nucleic acid blocks. The "leg" of the DNA walker could hybridize with a hairpin structure DNA named H1 and lead to the opening of it. And the newly exposed stem would interact with a primer. A strand exchange has happened with the assistance of polymerase and dNTPs, so that the "leg" has been displaced and the DNA walker could be pushed to move on the surface. But the nucleic acid blocks could increase steric hindrance and obstruct this process, which is similar to the behavior of human beings walking on craggy paths. Through changing these blocks, such as the structure, the amount, and the length of blocks, the movement of the DNA walker has been controlled. What's more, the results of its application for DNA detection are satisfactory. The limit of detection is 21.6 pM. Also, this method has been successfully applied in complex biological samples. Graphical abstract ᅟ.

A fluorometric turn-on aptasensor for mucin 1 based on signal amplification via a hybridization chain reaction and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots.

A fluorometric method is described for the determination of the tumor biomarker mucin 1 (MUC1). It is based on signal amplification of the hybridization chain reaction (HCR), and the interaction between a luminescent ruthenium(II) complex and CdZnTeS quantum dots (QDs). If MUC1 bind to the biotin-labeled aptamer, it will initiate HCR with hairpins H1 and H2 to form a long-range dsDNA. The long nucleic acid chains are then linked on the surface of streptavidin-modified magnetic microparticles (MMPs) through streptavidin-biotin interaction. The luminescent ruthenium(II) complex is then embedded in the long dsDNA linked to the MMPs. Hence, there is little Ru complex in the supernatant after magnetic separation, and the fluorescence of the CdZnTeS QDs (best measured at excitation/emission wavelengths of 350/530 nm) is only slightly quenched. In the absence of target, the fluorescence of the CdZnTeS QDs is strongly quenched. Fluorescence increases linearly in the 0.2-100 ng·mL-1 MUC1 concentration range, and the LOD is 0.13 ng·mL-1 (at S/N = 3). The method was applied to the determination of MUC1 in spiked human serum samples. Graphical abstract A fluorometric turn-on aptasensor for mucin 1 is described that is based on the interaction between a Ru(II) complex and quantum dots (QDs). The detection system includes biotin-labeled aptamer-H0, hairpins H1 and H2, streptavidin-modified magnetic microparticles (MMPs), Ru(bpy)2(dppx)2+ and CdZnTeS QDs.

Rox-DNA Functionalized Silicon Nanodots for Ratiometric Detection of Mercury Ions in Live Cells.

A ratiometric fluorescent sensor for mercury ions (Hg2+) has been constructed via covalent functionalization of silicon nanodot (SiND) with Hg2+-specific 6-carboxy-X-rhodamine (Rox)-tagged DNA. For the Rox-DNA functionalized SiND, the red fluorescence of Rox can be quenched by the blue-emitting SiND in the presence of Hg2+ due to structural change in DNA, which serves as the response signal. Meawhile, the fluorescence of SiND is insensitive to Hg2+ and acts as the reference signal. The wavelength difference in the optimal emission peak is as large as 190 nm between SiND (422 nm) and Rox (612 nm), which can efficaciously exclude the interference of the two emission peaks, and facilitates dual-color visualization of Hg2+ ions. The biofunctionalization of SiND improves the acid-base stability of SiND significantly, which is favorable for its application in the intracellular environment. Accordingly, a sensitive, simple, precise and rapid method for tracing Hg2+ was proposed. The limit of detection and precision of this method for Hg2+ was 9.2 nM and 8.8% (50 nM, n = 7), respectively. The increase of Hg2+ concentration in the range of 10-1500 nM was in accordance with linearly increase of the I422/ I612 ratio. As for practical application, the recoveries in spiked human urine and serum samples were in the range of 81-107%. Moreover, this fluorescent nanosensor was utilized to the ratiometric detection of Hg2+ in HeLa cells.

Simple construction of ratiometric fluorescent probe for the detection of dopamine and tyrosinase by the naked eye.

In this work, a simple and novel ratiometric fluorescence method based on ROX-DNA-functionalized CdZnTeS quantum dots (QDs) was developed for the detection of dopamine (DA) and tyrosinase (TYR). A ratiometric fluorescent probe was constructed by binding phosphorothioate DNA to the metal ions of QDs, which is a modification-free and low-cost method. DA was easily oxidized to DA quinone under the catalysis of TYR by dissolved O2, which effectively quenched the fluorescence of the QDs. Strong linear correlations were achieved for TYR in the range of 10.0-100.0 ng mL-1 and for DA in the range of 10.0-1000.0 nM. The limit of detection was estimated to be as low as 1.05 ng mL-1 for TYR and 1.93 nM for DA. Moreover, various colors were displayed in the course of detection, which could be observed by the naked eye. Therefore, an on-site and sensitive fluorescence method for the visual detection of DA and TYR can be developed. In addition, the findings revealed the potential applicability of the ratiometric fluorescent probe for the detection of DA and TYR in human serum. This ratiometric fluorescence method is not only sensitive and selective but also rapid and convenient for the detection of the analytes without sophisticated instrumentation.

Self-assembled protein-enzyme nanoflower-based fluorescent sensing for protein biomarker.

Multi-protein (or enzyme) conjugates play a vital role in biosensing due to the integrated function of each component, such as biological recognition and signal amplification. In this work, a green self-assembled method for the synthesis of multi-functional protein-enzyme nanoflowers has been developed, in which no chemical modification and coupling reaction is needed to fabricate the fluorescent signal probe. The self-assembled protein-enzyme conjugates streptavidin (SA) -ß-galactosidase (ß-Gal)-CaHPO4 nanoflowers load sufficient enzymes without damaging their activity, which meets the requirements of signal tags for biosensing. Through integrated multi-function of biorecognition (SA) and signal amplification (ß-Gal), the SA-ß-Gal-CaHPO4 hybrid nanoflower-based fluorescent sensor exhibited an ultrasensitive detection of protein biomarker alpha-fetoprotein (AFP), with limits of detection at the fM level. The presented self-assembled strategy can be extensively applied to develop on-demand protein-enzyme conjugates according to the specific requirements in a variety of applications including biosensors, bioimaging, and biomedicine. Graphical abstract A self-assembled method has been presented for the facile and green synthesis of SA-ß-Gal-CaHPO4 nanocomplexes with flower-like shape and high activity, and further employed as signal tag for fluorescent sensing of protein biomarker.