Click Chemistry-Mediated Particle Counting Sensing via Cu(II)-Polyglutamic Acid Coordination Chemistry and Enzymatic Reaction.

An electrical resistance-based particle counter (ERPC) with simple operation and high resolution has proved to be a promising biosensing toolkit, whereas amplification-free ERPC biosensors are incapable of analyzing trace small molecules due to their relatively low sensitivity. In this work, click chemistry-mediated particle counting sensing of small-molecule hazards in food samples with high sensitivity was developed. In this strategy, unbound alkyne-functionalized polystyrene microspheres were collected by magnetic separation from the copper-ion-mediated click reaction between alkyne-functionalized polystyrene microspheres and azido-functionalized magnetic beads, which could be used as signal probes for the readout. This click chemistry-mediated ERPC biosensor converts the detection of targets to the quantification of copper ions or ascorbic acid by performing competitive immunoassay-based coordination chemistry and enzymatic reaction, respectively. The sensitivity of the ERPC biosensor has been improved by an order of magnitude due to the signal amplification effects of click chemistry, coordination adsorption, and enzyme catalysis. Furthermore, because of the efficient separation and enrichment of immunomagnetic beads and the robustness of click chemistry, the interference from food matrixes and immunoassay is effectively reduced, and thus, our strategy is exceedingly suitable for detecting trace targets in complex samples.

Target-mediated competitive hybridization of hairpin probes for kanamycin detection based on exonuclease III cleavage and DNAzyme catalysis.

Based on aptamer recognition and target-mediated competitive hybridization of hairpin probes, we developed a fluorescence sensor for kanamycin (KAN) detection. The aptamer and KAN binding will open hairpin H1 to release the trigger DNA fragment, which can initiate the competitive hybridization between hairpins H2 and H3. Then, exonuclease III (Exo III) can cleave H2 and H3 to produce numerous DNA3 and DNA4. Through the synergetic hybridization among DNA1, DNA2, DNA3, and DNA4, an active Mg2+-DNAzyme can be formed. The cleavage reaction toward FAM-BHQ-modified DNA2 will produce a high fluorescence signal for KAN assay. Through Exo III-guided cleavage and Mg2+-DNAzyme-based catalysis, the sensor exhibits high sensitivity, with a detection limit of 3.1 fM. This method is robust and has been applied to the detection of KAN in milk and water samples with good accuracy and reliability. Our developed fluorescence sensor exhibits the advantages of simple operation, high sensitivity, and good robustness, which are beneficial for KAN detection in food samples.

Bioorthogonal Reactions Amplify Magnetic Nanoparticles Binding and Assembly for Ultrasensitive Magnetic Resonance Sensing.

Conventional transverse relaxation time (T2)-mediated magnetic resonance sensors (MRS) that utilizing the target-induces state change of magnetic nanoparticles (MNPs) mainly suffer from low sensitivity. Recent T2-MRS that based on target-induced amount change of MNPs can achieve a higher sensitivity, but these sensors can hardly accommodate small molecules. We herein develop an ultrasensitive T2-MRS that enable the detection of small molecules based on cascade bioorthogonal reactions (BRs)-realized MNPs binding and assembly. Benefiting from rapid and highly selective cascade BRs, a single small molecule target can not only increase MNPs binding but also assembly MNPs, which greatly amplifies T2 signal for sensing based on both the state and amount change of MNPs for the first time. Our strategy is capable of sensing chlorpyrifos with a liner range of 0.1 ng/mL to 1000 ng/mL. We justify the practicability of our assay by detecting chlorpyrifos in apple and cabbage samples, whose accuracy is higher than that of enzyme linked immunosorbent assay. Our assay provides a cascade BRs-mediated MRS that can greatly broaden the use of T2-based MRS for ultrasensitive sensing trace small molecules in complex samples.

Synergetic performance of isothermal amplification techniques and lateral flow approach for nucleic acid diagnostics.

The advancement in developing sensitive, rapid, and specific sensing tools is crucial in diagnostics and biotechnological applications. Although various isothermal amplification approaches exist for the detection and identification of nucleic acids, post-amplicon analysis is still based on traditional methods such as gel electrophoresis, colorimetry, turbidity, which could be non-specific and inconvenient. Thus, this review will first elaborate various isothermal amplification techniques (principle, merits, and demerits) and their potentials when combined with lateral flow approach for point-of-care nucleic acid diagnostics. Different methods for monitoring carryover contamination resulting from amplification product contamination will be discussed. Then, we will present recent advances in diagnostics with both target pre-amplification and CRISPR-Cas systems, which exhibit collateral cleavage of target nucleic acid and a reporter single strand nucleic acid within the vicinity. When the reporter is fluorophore-labeled, it provides a detectable signal by fluorescence or lateral flow biosensors. Lastly, we will discuss how CRISPR-Cas system based diagnostics could be more effective, affordable and portable for on-site detection.

A rapid and sensitive CRISPR/Cas12a based lateral flow biosensor for the detection of Epstein-Barr virus.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in the world, and several studies have associated Epstein-Barr virus (EBV) with NPC occurrence and development. EBV-PCR (polymerase chain reaction), in situ hybridization and immunoassays are the most common methods for NPC identification. However, these approaches have drawbacks, which include tedious procedures and false results. Therefore, a rapid, accurate, and sensitive clinical diagnostic method for the prognosis of EBV-related diseases is needed. In this study, we developed a simple and sensitive approach for EBV detection based on the combination of CRISPR-Cas12a and a lateral flow biosensor (LFB). Cas12a exhibits collateral cleavage propensity of both target DNA and any single-stranded(ss) DNA in the vicinity (herein referred to as a reporter). The LFB test line contained an ssDNA probe complementary to the reporter. In the presence of the target, Cas12a trans-cleaved the ssDNA reporter, which resulted in the inability of cleaved sequences to bind the LFB test line. With a PCR pre-amplification of the target (45 min), the assay achieved a sensitivity of 7.1 × 10-14 M (∼42 000 copies per µl) both in plasmid and plasmid-spiked samples. The assay attained a high specificity in the presence of various bacteria and applicability in EBV Burkitt's lymphoma serum samples. This method could be applied for the detection of EBV and other infectious diseases.

[Clinical features of coronavirus disease 2019 in children aged <18 years in Jiangxi, China: an analysis of 23 cases].

OBJECTIVE: To study the clinical features of coronavirus disease 2019 (COVID-19) in children aged <18 years. METHODS: A retrospective analysis was performed from the medical data of 23 children, aged from 3 months to 17 years and 8 months, who were diagnosed with COVID-19 in Jiangxi, China from January 21 to February 29, 2020. RESULTS: Of the 23 children with COVID-19, 17 had family aggregation. Three children (13%) had asymptomatic infection, 6 (26%) had mild type, and 14 (61%) had common type. Among these 23 children, 16 (70%) had fever, 11 (48%) had cough, 8 (35%) had fever and cough, and 8 (35%) had wet rales in the lungs. The period from disease onset or the first nucleic acid-positive detection of SARS-CoV-2 to the virus nucleic acid negative conversion was 6-24 days (median 12 days). Of the 23 children, 3 had a reduction in total leukocyte count, 2 had a reduction in lymphocytes, 2 had an increase in C-reactive protein, and 2 had an increase in D-dimer. Abnormal pulmonary CT findings were observed in 12 children, among whom 9 had patchy ground-glass opacities in both lungs. All 23 children received antiviral therapy and were recovered. CONCLUSIONS: COVID-19 in children aged <18 years often occurs with family aggregation, with no specific clinical manifestation and laboratory examination results. Most of these children have mild symptoms and a good prognosis. Epidemiological history is of particular importance in the diagnosis of COVID-19 in children aged <18 years.

Highly Sensitive Aptasensor for Trace Arsenic(III) Detection Using DNAzyme as the Biocatalytic Amplifier.

A highly sensitive fluorescence biosensing system was designed for the detection of trace amounts of arsenic(III) (As3+) based on target-triggered successive signal amplification strategy. The specific recognition between the target As3+ and the aptamer sequence results in the release of the blocking DNA to trigger the subsequent signal amplification steps. Exonuclease III (Exo III)-mediated DNA recycling digest process is introduced into the sensing system to generate numerous Mg2+-dependent DNAzymes. After magnetic separation, the active DNAzyme with multiple turnovers could catalyze the continuous cleavage of the fluorophore-quencher-functionalized substrate strands, thus yielding a significantly amplified fluorescence signal for target detection. Due to the synergetic signal amplification of Exo III and DNAzyme, the fluorescent biosensor exhibits ultrasensitivity for As3+ monitoring, with a detection limit of 2 pM. Our established biosensor also displays excellent selectivity toward the target As3+ and has been successfully applied to the determination of As3+ in water samples with satisfactory accuracy. This sensing platform can be developed as a universal approach for the fast, sensitive, and accurate detection of aptamer-binding molecules.

Accurate Clinical Diagnosis of Liver Cancer Based on Simultaneous Detection of Ternary Specific Antigens by Magnetic Induced Mixing Surface-Enhanced Raman Scattering Emissions.

Establishing an accurate, simple, and rapid serodiagnosis method aiming for specific cancer antigens is critically important for the clinical diagnosis, therapy, and prognostication of cancer. Currently, surface-enhanced Raman scattering (SERS) readout techniques challenge fluorescent-based detection methods in terms of both optical stability and more importantly multiple detection capability, which become more desirable for clinical diagnostics. We thus started using an interference-free mixing SERS emission (m-SERS) readout to simultaneously indicate, for the first time, three specific liver cancer antigens, including α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and ferritin (FER), even in one clinical serum sample. Here, three triple bonds (C≡N and C≡C) coded SERS tags contribute separate SERS emissions located at 2105, 2159, and 2227 cm-1, respectively; must have one-to-one correspondence from AFP, to FER, to CEA, In the process of detection, the mature double antibody sandwich allows the formation of microscale core-satellite assembly structure between a magnetic bead (MB) and single SERS tags, and therefore a pure and single SERS emission can be observed under the routine excitation laser spot. Because of the action of magnetic force, the uniform 3D packing of SERS tags absorbed MBs will in contrast generate a so-called m-SERS signals. With the help of enrichment and separation by MBs, the proposed m-SERS immunoassay provides an extremely rapid, sensitive, and accurate solution for multiplex detection of antigens or other biomarkers. Herein, the limit of detection (LOD) for simultaneous m-SERS detection of AFP, CEA, and FER was 0.15, 20, and 4 pg/mL, respectively. As expected for 39 clinical serum samples, simultaneous detection of ternary specific antigens can significantly improve the accuracy of liver cancer diagnosis.

Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 ß-Agonists and Analogues.

Immunoassay methods are important for monitoring ß-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various ß-agonist chemicals that are possibly present in food. In this study, through the use of an R-(-)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 ß-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1', dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2' or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) was established with an ideal recovery ranging from 81.8 to 118.3% based on the obtained antibody. With a good agreement to the HPLC/MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of ß-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

[Application of gold nanoparticles-based lateral flow strip for dengue virus detection based on strand-displacement amplification].

OBJECTIVE: To investigate the rapid and accurate method for the detection of dengue virus (DENV) by using nicking enzyme assisted strand-displacement amplification (SDA) combined with gold nanoparticles-based lateral flow strip. METHODS: Total RNA of the virus was extracted by using magnetic beads method and transcribed to cDNA for SDA detection system. Nicking enzyme-assisted method was used for detecting DENV, and agarose gel electrophoresis was used for analyzing the sensitivity of SDA amplification products. A gold nanoparticles-based lateral flow strip was developed based on the principle of nucleic acid base complementary pairing to design the test line and control line. The gold particles were prepared by using sodium citrate reduction method for gold nanoparticles-based lateral flow strip construction. RESULTS: The sensitivity of the SDA method was 10 fmol/L, and the sensitivity of gold nanoparticles-based lateral flow strip based on SDA method was also 10 fmol/L. In a linear range from 10 fmol/L to 1012 fmol/L, the corresponding linear correlation coefficient (R2) of DENV was 0.98. The specificity of nanoparticles-based lateral flow strip based on SDA for DENV detection was high, which was no crossing with other control groups. CONCLUSIONS: A gold nanoparticles-based lateral flow strip based on SDA method for DENV detection has been established, which is convenient, fast, and the result is visible to naked eyes.

Lateral flow biosensor for multiplex detection of nitrofuran metabolites based on functionalized magnetic beads.

The use of potential mutagenic nitrofuran antibiotic in food animal production has been banned world-wide. Common methods for nitrofuran detection involve complex extraction procedures. In the present study, magnetic beads functionalized with antibody against nitrofuran derivative were used as both the extraction and color developing media in lateral flow biosensor. Derivatization reagent carboxybenzaldehyde is firstly modified with ractopamine. After reaction with nitrofuran metabolites, the resultant molecule has two functional groups: the metabolite moiety and the ractopamine moiety. Metabolite moiety is captured by the antibody that is coated on magnetic beads. This duplex is then loaded onto biosensor and ractopamine moiety is further captured by the antibody immobilized on the test zone of nitrocellulose membrane. Without tedious organic reagent-based extraction procedure, this biosensor was capable of visually detecting four metabolites simultaneously with a detection limit of 0.1 µg/L. No cross-reactivity was observed in the presence of 50 µg/L interferential components. Graphical abstract Derivatization of nitrofuran metabolites (AHD, AOZ, SEM, or AMOZ) and LFA detection of the derivative products.

Colorimetric detection of copper(II) ion using click chemistry and hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme.

G-quadruplex-forming sequence can be formed through a copper(I) ion (Cu(+))-catalyzed click chemistry between azide- and alkyne-modified short G-rich sequences in aqueous solution, eliminating immobilization and washing steps of conventional assays. The source for Cu(+) was generated from the reduction of Cu(2+) with the reductant of sodium ascorbate. In the presence of hemin and K(+), the self-assembly of hemin/G-quadruplex structure has the activity of horseradish peroxidase (HRP), which can catalyze its colorless substrate tetrazmethyl benzidine (TMB) into a colored product. Hence, the concentration of Cu(2+) can be evaluated visually for qualitative analysis according to the color change of the solution, and the optical density (OD) value of the resulting solution at 450 nm was also recorded using a microplate reader for quantitative analysis.

Microfluidic formulation of food additives-loaded nanoparticles for antioxidation.

Browning has many important implications with nutrition and the shelf life of foods. Mitigating browning is of particular interest in food chemistry. The addition of antioxidants has been a common strategy to extend shelf life of drug and food products. In this work, we report a microfluidic technology for encapsulation of three common food additives (potassium metathionite (PMS), curcumin (CCM), and ß-carotene (ß-Car)) into nano-formulations using low-cost and readily available materials such as shellac. The food additives encapsulated nanoparticles provide a microenvironment that can prevent oxidation during daily storage. The results showed that the produced nanoparticles had a narrow size distribution with an average size of around 100 nm, were stable at conventional storage conditions (4 ºC) for 18 weeks, and had sustained release ability at 37 ºC, pH= 7.8, 160 rpm. In addition, further experiments showed that the formulation of hydrophobic additives, such as CCM and ß-Car did not only improve their bioavailability but also allowed for the encapsulation of a combination of ingredients. In addition, the antioxidants loaded nanoparticles demonstrated good biocompatibility, low toxicity to human cells. The longer release time of encapsulated food additives increases shelf life of foods and enhances consumer purchase preferences, which not only saves costs but also reduces waste. In summary, this study shows that such antioxidant-loaded nanoparticles provide a promising strategy in extending the shelf life of food products.

An enhanced strip biosensor for rapid and sensitive detection of histone methylation.

Histone methylation is a crucial epigenetic modification of chromosomes. In this work, we describe an enhanced strip biosensor using oligonucleotide-functionalized gold nanoparticles as an enhancer probe (AuNP-DNA) for rapid and sensitive detection of histone methylation. In conventional strip biosensor, methylated histone is captured on the test zone through the formation of antibody/methylated histone/antibody-labeled AuNP sandwich structures. Whereas, in the enhanced strip biosensor, the AuNPs in the sandwich structures are dual labeled with an antibody and another oligonucleotide (c-DNA). The sequence of the c-DNA is complementary to the oligonucleotide on the enhancer probe. The enhancer probe, AuNP-DNA, hybridizes with the c-DNA on the dual labeled AuNPs, and the color intensity of the red band on the test zone is then enhanced dramatically. The enhanced strip biosensor has been used for the visual detection of trimethylated lysine 9 of histone H3 (H3K9me3) in 20 ng of histone extract from HeLa cells within 15 min. The detection limit is 10-fold and 15-fold lower than the conventional strip biosensor and Western blot, respectively.

A lateral flow biosensor for the detection of human pluripotent stem cells.

A lateral flow biosensor based on immunoassay has been developed for the detection of human stem cells for the first time. Antibody specific for a human stem cell surface antigen, SSEA-4, is coated onto gold nanoparticles, whereas antibody against another human pluripotent stem cell surface antigen, SSEA-3, is immobilized on the test zone of the NC membrane. Target cells bind to the antibody coated on the gold nanoparticles to form nanoparticles-stem cell complexes, and the complexes are then captured by another antibody immobilized on the test zone to form a red line for visual detection. This biosensor has been successfully applied to human embryonic stem cells and induced pluripotent stem cells. It is capable of detecting a minimum of 10,000 human embryonic stem cells by the naked eye and 7000 cells with a portable strip reader within 20 min. This approach has also shown excellent specificity to distinguish other types of cells. The biosensor shows great promise for specific and handy detection of human pluripotent stem cells.

An enzyme-free and label-free assay for copper(II) ion detection based on self-assembled DNA concatamers and Sybr Green I.

An enzyme-free and label-free fluorescence turn on biosensor for amplified copper(II) ion (Cu(2+)) detection has been constructed based on self-assembled DNA concatamers and Sybr Green I. This assay is simple, inexpensive and sensitive, enabling quantitative detection of as low as 12.8 pM Cu(2+).

Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction.

We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.

Programmable DNA biocomputing circuits for rapid and intelligent screening of SARS-CoV-2 variants.

The frequent emergence of SARS-CoV-2 variants increased viral transmissibility and reduced protection afforded by vaccines. The rapid, multichannel, and intelligent screening of variants is critical to minimizing community transmissions. DNA molecular logic gates have attracted wide attention in recent years due to the powerful information processing capabilities and molecular data biocomputing functions. In this work, some molecular switches (MSs) were connected with each other to implement arbitrary binary functions by emulating the threshold switching of MOS transistors and the decision tree model. Using specific sequences of different SARS-CoV-2 variants as inputs, the MSs net was used to build several molecular biocomputing circuits, including NOT, AND, OR, INHIBIT, XOR, half adder, half subtractor, full adder, and full subtractor. Four fluorophores (FAM, Cy3, ROX, and Cy5) were employed in the logic systems to realize the multichannel monitoring of the logic operation results. The logic response is fast and can be finished with 10 min, which facilitates the rapid wide-population screening for SARS-CoV-2 variants. Importantly, the logic results can be directly observed by the naked eye under a portable UV lamp, thus providing a simple and intelligent method to enable high-frequency point-of-care diagnostics, particularly in low-resource communities.

Construction of molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly and CRISPR-Cas12a.

We successfully constructed several molecular logic gates using heavy metal ions as inputs based on catalytic hairpin assembly (CHA) and CRISPR-Cas12a. The corresponding DNAzymes were used to recognize heavy metal ions (Hg2+, Cd2+, Pb2+, and Mn2+). The specific cleavage between heavy metal ions and DNAzymes leads to the release of the trigger DNA, which can be used to activate CHA through logic computation. The CHA-generated DNA duplexes contain the protospacer adjacent motifs (PAM) sequence, which can be distinguished by CRISPR-Cas12a. The hybridization interactions between the duplexes and gRNA will activate the trans-cleavage capability of Cas12a, which can cleave the single-stranded DNA (ssDNA) reporter. The separation of the fluorescence group and quench group in ssDNA will generate a high fluorescence signal for readout. Using Hg2+ and Cd2+ as the two inputs, several basic logic gates were constructed, including OR, AND, and INHIBT. Using Hg2+, Cd2+, Pb2+, and Mn2+ as the four inputs, cascaded logic gates were further fabricated. With the advantages of scalability, versatility, and logic computing capability, our proposed molecular logic gates can provide an intelligent sensing system for heavy metal ions monitoring.

In Situ Deposition of Gold Nanoparticles and L-Cysteine on Screen-Printed Carbon Electrode for Rapid Electrochemical Determination of As(III) in Water and Tea.

In this study, a screen-printed carbon electrode (SPCE) based on in situ deposition modification was developed for the sensitive, rapid, easy and convenient determination of As(III) in water and tea by linear sweep anodic stripping voltammetry (LSASV). The screen-printed carbon electrodes were placed in a solution consisting of As(III) solution, chlorauric acid and L-cysteine. Under certain electrical potential, the chloroauric acid was reduced to gold nanoparticles (AuNPs) on the SPCE. L-cysteine was self-assembled onto AuNPs and promoted the enrichment of As(III), thus enhancing the determination specificity and sensitivity of As(III). The method achieved a limit of determination (LOD) of 0.91 ppb (µg L-1), a linear range of 1~200 µg L-1, an inter-assay coefficient of variation of 5.3% and good specificity. The developed method was successfully applied to the determination of As(III) in tap water and tea samples, with a recovery rate of 93.8%~105.4%, and further validated by inductively coupled plasma mass spectrometry (ICP-MS). The developed method is rapid, convenient and accurate, holding great promise in the on-site determination of As(III) in tap water and tea leaves, and it can be extended to the detection of other samples.