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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.

Nervous necrosis virus (NNV) has infected more than 50 fish species worldwide, and has caused serious economic losses in the aquaculture industries. However, there is no effective antiviral therapy. The development of a rapid and accurate point-of-care diagnostic method for the prevention and control of NNV infection is urgently required. Commonly used methods for NNV detection include the cell culture-based assay, antibody-based assay and polymerase chain reaction (PCR)-based assay. However, these methods have disadvantages as they are time-consuming and complex. In the present study, we developed a simple and sensitive aptamer-based lateral flow biosensor (LFB) method for the rapid detection of red-spotted grouper nervous necrosis virus (RGNNV). An aptamer is a single-stranded nucleotide, which can specifically bind to the target and has many advantages. Based on a previously selected aptamer, which specifically bound to the coat protein of RGNNV (RGNNV-CP), two modified aptamers were used in this study. One aptamer was used for magnetic bead enrichment and the other was used for isothermal strand displacement amplification (SDA). After amplification, the product was further tested by the LFB, and the detection results were observed by the naked eye within 5 min with high specificity and sensitivity. The LFB method could detect RGNNV-CP protein as low as 5 ng/mL or 5 × 103 RGNNV-infected GB (grouper brain) cells. Overall, it is the first application of a LFB combined with aptamer in the rapid diagnosis of virus from aquatic animals, which provides a new option for virus detection in aquaculture.

We have developed an equipment-free biosensor for visual pesticide residue (acetamiprid) detection with high sensitivity and selectivity. The target acetamiprid associates with an aptamer sequence and releases trigger DNA to initiate the cyclic signal amplification process. An ingeniously designed hairpin DNA probe was employed as the sensing element, which contains the same sequence as the trigger DNA in the stem part of the hairpin and a G-rich sequence in the loop part of the hairpin. An exonuclease III (Exo III)-mediated target recycling strategy was utilized to achieve quadratic amplification. This two-step cyclic signal amplification results in the generation of numerous G-quadruplex DNAzymes that can catalyze the H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate a colored signal readout, thus providing the amplified colorimetric detection of the target. This assay is ultrasensitive, enabling the visual detection of acetamiprid at concentrations as low as 10 pM without instrumentation. Our proposed sensing system also displays high selectivity and can be applied to determine acetamiprid residues in food samples. Moreover, the assay does not involve any chemical modification of DNA, which is simple and low-cost. This sensing platform provides a promising approach for on-site detection of target molecules in resource-constrained regions because of the easy and straightforward readout of results without the use of sophisticated apparatus.

An enzyme-free DNA circuit was designed for the amplified detection of Cd2+ based on hairpin probe-mediated toehold binding and branch migration. A Cd2+-specific aptamer was used to recognize Cd2+ and a G-quadruplex was used to report the detection signal. The assay is sensitive, with a detection limit of 5 pM.

Bacterial infections may lead to diverse acute or chronic diseases (e.g., inflammation, sepsis and cancer). New antibiotics against bacteria are rarely discovered in recent years, which necessitates the exploration of new antibacterial agents. Engineered nanomaterials (ENMs) have been extensively studied for antibacterial use because of their long lasting killing effects in wide spectra of bacteria. Graphene oxide (GO) is one of the most widely studied ENMs and exhibit strong bactericidal effects. The physicochemical properties of GO play important roles in bacterial killing by triggering a cascade of toxic events. Many studies have explored the signaling pathways of GO in bacteria. Although molecular initiating events (MIEs) of GO in bacteria dominate its killing efficiency as well as toxicity mechanisms, they have been rarely reviewed. In this report, we discussed the structure-activity relationships (SARs) involved in GO-induced bacterial killing and the MIEs including redox reaction with biomolecules, mechanical destruction of membranes and catalysis of extracellular metabolites. Furthermore, we summarized the clinical or commercial applications of GO-based antibacterial products and discussed their biosafety in mammal. Finally, we reviewed the remaining challenges in GO for antibacterial applications, which may offer new insights for the development of nano antibacterial studies.

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 sensiti...

CRISPR/Cas systems have displayed remarkable potential in developing novel biosensing applications for nucleic acid detection owing to the collateral cleavage activity of Cas effector proteins (Cas12, Cas13, etc.). Despite tremendous progress in recent years, the existing CRISPR/Cas based biosensing platforms have several limitations, including reliance on proper amplification methods, expensive fluorescence detection equipment, or lateral flow biosensor (LFB). Herein, we report a simple, inexpensive, and ultrasensitive DNA probe based LFB with C RISPR/Cas and loop-mediated I sothermal A mplification (namely CIA). The concept behind this approach is a non-detectable test line on the LFB when the Cas effector protein collaterally cleaves the cognate target and an ssDNA reporter sequence. The CIA based LFB can detect as low as a single copy cloned Pseudomonas aeruginosa acyltransferase gene, 1 cfu/ml plasmid containing E. coli DH5α pure cultures, as well as clinical samples without DNA extraction/purification or advanced apparatuses. No cross-reactivity with other non-target bacteria was observed. The naked eye result readout was obtained in 15 min of LAMP amplification, 30 min of Cas12 reaction, and 5 min of LFB readout. This platform is robust and of low cost for on-site testing.

The rapid determination of human osteopontin (OPN) protein, a potential cancer biomarker, holds substantial promise for point-of-care diagnostics and biomedical applications. To date, most reported platforms for OPN detection are apparatus-dependent, time-consuming, and expensive. Herein, we established a lateral flow biosensor (LFB) for OPN detection. A biotinylated aptamer was used for OPN pre-capture from samples, an antibody for OPN was immobilized on the test line for a second specific target identification, and streptavidin-modified gold nanoparticles were sprayed on the conjugation pad for color detection. This LFB achieved as low as 0.1 ng mL−1 OPN sensitivity with a good dynamic detection between 10 and 500 ng mL−1 within 5 min. Intriguingly, the LFB allowed a qualitative and semi-quantitative detection of OPN in serum at clinically cut-off levels as in cancer patients, and can discriminate OPN from interfering proteins with high specificity. Thus, it is a promising alterative approach for point-of-care OPN screening and detection.

Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful, however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR&ndash, Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 &times, 10&minus, 15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices, thus, it can be applied to virus monitoring and food samples detection.

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+ an...

As an ubiquitous heavy metal pollutant, cadmium ion (Cd2+) is detrimental to food and human health even at low concentrations. Conventional methods require costly instruments and cannot meet the requirements of on-site analysis. Here we report the use of a personal glucose meter (PGM) as the point-of-use (POU) device for portable and quantitative detection of Cd2+. The specific recognition between the aptamer and Cd2+ trigger the recycling signal amplification process by exonuclease III (Exo III). After successive hybridization and cleavage reactions, numerous single-stranded DNA were liberated on the surface of the magnetic bead. An invertase-conjugated DNA that is complementary to the single-stranded DNA is introduced into the sensing system. After magnetic separation, the invertase conjugates hydrolyze sucrose into glucose, thus establishing direct conversion of Cd2+ concentration to glucose amount, which can be directly quantified by a PGM. Thanks to the synergistic signal amplification of Exo III and invertase, the POU device greatly improves the sensitivity for Cd2+ analysis, with a detection limit of 5 p.M. With the advantages of portability, cost-effectiveness, wide availability, and ease of use, the PGM-based detector has the potential to be used by the public as a routine tool for reliable and quantitative detection of Cd2+.

A wash-free and label-free colorimetric biosensor for the amplified detection of aflatoxin B1 (AFB1) has been constructed by the integration of an ingenious hairpin DNA probe with exonuclease III (Exo III)-assisted signal amplification. The presence of the AFB1 activates the continuous cleavage reactions by Exo III toward a hairpin probe, resulting in the autonomous accumulation of numerous free G-quadruplex sequences, which can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to produce a colorimetric response. The naked-eye biosensor is ultrasensitive, enabling the visual detection of trace amounts of AFB1 as low as 1 pM without instrumentation. The sensor is robust and can work even when challenged with complex sample matrices such as peanut samples. With the advantages of simple operation, wash-free and label-free format, visible and intuitive output, and low cost, the naked-eye based colorimetric biosensor is expected to have potential applications for in-field detection of AFB1.

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells.

Immunoassay methods are important for monitoring β-agonists illegally used for reducing animal fat deposition in livestock. However, there is no simultaneous screening surveillance immunoassay for detecting various β-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 β-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 β-agonists in urine. This investigation will contribute to the rational design and control of the immunoassay specificity.

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.

Copper(II) ions (Cu2+) at a high concentration are harmful to human health. Herein a simple and enzyme-free lateral flow biosensor for the rapid detection of Cu2+ based on copper(I) ion (Cu+)-catalyzed click chemistry has been constructed for the first time. In the presence of sodium ascorbate, Cu2+ was reduced to Cu+, which could catalyze the cycloaddition between azide-DNA and alkyne/biotin-DNA in aqueous solution. The ligated DNA product could then be immobilized onto the test zone of the lateral flow biosensor to form a red band which could be unambiguously read by the naked eye. Taking advantage of the optical properties of gold nanoparticles (AuNPs) and high efficiency and selectivity of Cu+-catalyzed click chemistry, this assay enabled the visual detection of Cu2+ as low as 100 nM with excellent specificity. In comparison with conventional methods, this biosensor is more simple to operate and more cost-effective to use, and therefore has great potential in point-of-care diagnosis and environmental monitoring.

The analysis of intracellular ATP can reveal the response of cells to different treatments and is important for individualized medicine. In the present study, we developed a cell penetrating peptides (CPPs) tagged luciferase (TAT-LUC) for tumor chemosensitivity assay. The activity of recombinant TAT-LUC was evaluated using ATP standard solution and tumor cells. This recombinant TAT-LUC was then used for the analysis of sensitivity index (SI) of four strains of tumor cells. The results showed that TAT-LUC could detect less than 10 nM extracellular ATP with a strong correlation between the luminescence intensity and the ATP content (R2 = 0.994). Without cell lysis, the detection limit for intracellular ATP analysis was 40 tumor cells. Furthermore, chemosensitivity of four strains of tumor cells (Skov-3/DDP, A549/DDP, MDA-MB-231, Huh-7) was determined by this assay successfully. The cell penetration ability of TAT-LUC enables the assay not only to reflect drug resistance of tumor cells real-timely but also to minimize the test time, which can be a valuable aid for personalized cancer chemotherapy.

Utilizing G-quadruplex as the signal report probe, an ultrasensitive and label-free autocatalytic DNA circuit for Hg2+ detection on the basis of exonuclease III (Exo III)-assisted cascade signal amplification has been proposed. In the absence of Hg2+, the hairpin A and the DNA1 cannot hybridize due to the thymine-thymine (T-T) mismatches. Therefore, hairpin probes with the 3′-protruding terminus can be resistant to Exo III digestion, preventing the G-rich sequence to be released. In the presence of Hg2+, the combination of the DNA1 with the 3′ end-extruding hairpin A via T-Hg2+-T coordination chemistry triggers the digestion reaction of Exo III, leading to the release of the DNA1 and the sequence with domains c, d, and e. Both of the DNA1 and the sequence with domains c, d, and e can combine with other hairpin probes and activate another round of the cleavage reaction. The produced G-rich sequence can form G-quadruplex structure by binding with N-Methyl mesoporphyrin IX (NMM). The biosensor exhibits excellent selectivity and high sensitivity for Hg2+. The linear range of this biosensor is from 10 fM to 100 nM, and the linear equation can be expressed as: F610 = 1.3 × 105 Lg C + 7.40 × 104 (R2 = 0.998), in which F610 is the fluorescence intensity at 610 nm, C represents the Hg2+ concentrations, and Lg is the logarithm of 10. The detection limit is 10 fM. The biosensor is robust and can be applied to the detection of Hg2+ in water samples. By substituting the target-recognition elements, this sensing system can also be used for the detection of other metal ions.

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.

A universal biosensor for the portable and quantitative detection of transcription factors has been constructed using a commercially available glucometer as the sensing platform. With the specific protein-binding DNA and antibody as the recognition elements, invertase as the linker, and glucometer as the transducer, quantitative detection was achieved via target-induced capturing of invertase conjugates on magnetic beads, thereby transforming the concentration of the target in the sample into glucose through invertase-catalyzed hydrolysis of sucrose. In comparison with laboratory-based instruments or customized devices, the glucometer-based biosensor has the significant advantages of low cost, compact size, wide accessibility, and ease of use, making it as convenient for use at home as in the field. As a proof of concept, Oct4, an important transcription factor for the regulation of the process of embryonic stem cells differentiation, was used as the model target. Using the proposed point-of-care strategy, Oct4 can be quantified in the range from 0.05 to 25 ng mL−1 with a detection limit of 0.05 ng mL−1, which is comparable to the commercial Oct4 test kits. The glucometer-based biosensor is robust and can be used directly to measure the transcription factor activities in crude cell lysate with excellent selectivity. It is expected that this assay principle can be directed towards other DNA-binding transcription factors by simply changing the binding site sequence and the corresponding antibody.

An autonomous thymine rich DNA machine as an amplification unit was developed for the sensitive detection of mercury ions with high specificity. Combined with a lateral flow biosensor, the amplified signal of Hg2+ can be read out by the naked eye with a detection limit of 5 nM.

In this communication, we report a rapid and cost-efficient assay for the detection of S. enteritidis. Specific aptamers with fluorescence labeled S. enteritidis are absorbed into graphene oxide, and the fluorescence is quenched owing to the fluorescence quenching ability of graphene oxide. In the presence of S. enteritidis, the aptamer would release from the graphene oxide to obtain a significant fluorescence recovery. This aptasensor can detect as low as 40 CFU mL−1 of S. enteritidis in 30 min, and can fulfill the demand of multiplex detection. The cost-effective, rapid and simple aptasensor offers a promising method for food-borne pathogen monitoring during food processing.

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 series of enzyme-amplified electronic logic gates (OR, AND, NOR, and NAND) for one-spot simultaneous monitoring of small molecules and proteins has been constructed at the molecular level. This simple but universal system is based on target-induced self-assembly of split aptamer fragments or target-induced conformational changes of intact aptamers. For the OR and AND logic operations, the split aptamer fragments were used as the molecular recognition components, while for the NOR and NAND logic operations, the intact aptamers were used as the molecular recognition components. Using ATP and thrombin as inputs, the split/intact aptamers as molecular recognition elements, biotin as a tracer, and SA–HR as a reporter molecule, the electronic logic operations can be easily realized by generating amplified current signals as outputs. The logic system is robust and can be applied to human serum samples with excellent selectivity. Importantly, the reversibility of these logic gates makes the electronic system feasible to perform the set–reset function. Our work not only provides a “smart” and flexible logic platform for ATP and thrombin sensing, but also can be expanded for other ligands assay, such as adenosine monophosphate (AMP), theophylline, and cocaine, by rationally splitting their aptamer sequences into two fragments.

A lateral flow biosensor (LFB) is introduced for the detection of single nucleotide polymorphisms (SNPs). The assay is composed of two steps: circular strand displacement reaction and lateral flow biosensor detection. In step 1, the nucleotide at SNP site is recognized by T4 DNA ligase and the signal is amplified by strand displacement DNA polymerase, which can be accomplished at a constant temperature. In step 2, the reaction product of step 1 is detected by a lateral flow biosensor, which is a rapid and cost effective tool for nuclei acid detection. Comparing with conventional methods, it requires no complicated machines. It is suitable for the use of point of care diagnostics. Therefore, this simple, cost effective, robust, and promising LFB detection method of SNP has great potential for the detection of genetic diseases, personalized medicine, cancer related mutations, and drug-resistant mutations of infectious agents.

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.

A fast and reliable sensing platform has been developed for the detection of mycotoxin ochratoxin A (OTA) based on a target-induced structure-switching signaling aptamer. In the absence of target, a fluorescein-labeled OTA aptamer hybridizes to a complementary DNA strand containing a quencher moiety, bringing the fluorophore and the quencher into close proximity for highly efficient fluorescence quenching. Upon OTA addition, a conformational change in the aptamer releases the quencher-containing DNA strand, generating a strong concentration-dependent fluorescent signal. Using this technique, the entire detection and analysis process of OTA can be completed within 1 min. Under optimized assay conditions, a wide linear detection range (from 1 to 100 ng/mL) was achieved with a detection limit down to 0.8 ng/mL. Additionally, the proposed assay system exhibited high selectivity for OTA against other mycotoxins (aflatoxin B 1 and zearalenone) and limited interference from the structural analog ochratoxin B. The biosensor was also applied to a non-contaminated corn material spiked with a dilution series of OTA, obtaining recoveries from 83% to 106%. Utilization of the proposed biosensor for quantitative determination of mycotoxins in food samples may provide significant improvements in quality control of food safety through a simple, rapid, and sensitive testing system for agricultural products monitoring.

A lateral flow biosensor based on an immuno-chromatographic assay has been developed for the detection of DNA-binding proteins. The biosensor is composed of four parts: a sample pad, a conjugate pad, a strip of nitrocellulose membrane and an absorbent pad. A DNA probe containing a specific protein binding consensus sequence is coated onto gold nanoparticles, while an antibody against the DNA-binding protein is immobilized onto a test zone of the nitrocellulose membrane. The target protein binds to the protein binding DNA sequence that is coated on the gold nanoparticles to form nanoparticle-DNA-protein complexes, and the complexes are then captured by the antibody immobilized on the test zone to form a red line for visual detection of the target protein. This biosensor was successfully applied to a DNA-binding protein, c-jun, and the developed biosensor allows for the rapid detection of down to 0.2 footprint unit of c-jun protein within 10 min. This biosensor was verified using HeLa cells and it visually detected c-jun activity in 100 μg of crude cell lysate protein. The antibody against c-jun used in the biosensor can distinguish c-jun from other nonspecific proteins, with high specificity.

Vascular endothelial growth factor (VEGF) is one of the best characterized angiogenic factors controlling placental angiogenesis; however, how VEGF regulates placental angiogenesis has not yet completely understood. In this study, we found that all the components of assembling a functional NADPH oxidase (NOX2, p22phox, p47phox, p67phox, and Rac1) are expressed in ovine fetoplacental artery endothelial cells (oFPAECs) in vitro and ex vivo. Treatment with VEGF (10 ng/ml) rapidly and transiently activated Rac1 in oFPAECs in vitro and increased Rac1 association with p67phox in 5 min. Intracellular superoxide formation began to significantly increase after 25–30 min of VEGF stimulation, which was mediated by both VEGFR1 and VEGFR2. VEGF also stimulated oFPAE cell proliferation and migration and enhanced the formation of tube-like structures on Matrigel matrix. In oFAPEC transfected with specific Rac1 small interfering RNA (siRNA, 40 nm), VEGF-induced intracellular superoxide formation was completely abrogated in association with a 78% reduction of endogenous Rac1. In oFPAE cells transfected with the specific Rac1 siRNA, but not with transfection reagent alone or scrambled control siRNA, VEGF-induced cell proliferation, migration, and tube-like structure formation were dramatically inhibited. Pretreatment of an NADPH oxidase inhibitor apocynin also abrogates the VEGF-stimulated intracellular superoxide production and DNA synthesis in oFPAECs. Taken together, our results demonstrated that a Rac1/Nox2-based NADPH oxidase system is present in placental endothelial cells. This NADPH oxidase system appears to generate the second messenger superoxide that plays a critical role in the signaling control of the VEGF-induced placental angiogenesis.

Vanillin (4-hydroxy-3-methoxybenzaldehyde), a food additive with rich milk flavor, is commonly used in the food, beverage and cosmetic industries. However, excessive consumption of vanillin may cause liver and kidney damage. Therefore, methods for detecting and controlling the level of vanillin in food, especially in infant powder, have important practical significance. In this study, we established a colorimetric assay for vanillin detection. The detection was performed under high-temperature and acidic conditions, which can induce the reaction of the aldehyde group of vanillin with the amino group of o-toluidine. The resulting product had a maximum absorption at 363 nm, which was quantified by a UV spectrophotometer. This assay had a limit of detection (LOD) of 1 pg mL-1 and a linear range between 1 μg mL-1 and 100 μg mL-1. The average recoveries at three spiked levels were in the range from 91.1% to 101.6% with a relative standard deviation (RSD) of 4.62% ~ 7.27%.

We report an aptamer-nanoparticle strip biosensor (ANSB) for the rapid, specific, sensitive and low-cost detection of circulating cancer cells. Known for their high specificity and affinity, aptamers were first selected from live cells by the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. When next combined with the unique optical properties of gold nanoparticles (Au-NPs), ANSBs were prepared on a lateral flow device. Ramos cells were used as a model target cell to demonstrate proof of principle. Under optimal conditions, the ANSB was capable of detecting a minimum of 4000 Ramos cells without instrumentation (visual judgment) and 800 Ramos cells with a portable strip reader within 15 minutes. Importantly, ANSB has successfully detected Ramos cells in human blood, thus providing a rapid, sensitive and low-cost quantitative tool for the detection of circulating cancer cells. ANSB therefore shows great promise for in-field and point-of-care cancer diagnosis and therapy.

A novel glucometer biosensor was developed for the quantitative detection of mercury ions (Hg2+) based on glucose-loaded DNA-gated mesoporous silica nanoparticles (MSNs).

Summary Alpha-fodrin, an intracellular organ-specific cytoskeleton protein, was identified recently as an autoantigen associated with Sicca- and Sjögren's syndrome (SS). Identification of the antigenic determinants of α-fodrin is a prerequisite to developing highly sensitive and specific anti-α-fodrin antibodies, which provides potential means for the diagnosis of primary Sjögren's syndrome (pSS) in patients. Based on the structure and predicted antigenic sites of α-fodrin protein with 560 amino acids (α-fodrin 560), we prepared a set of overlapping recombinant protein fragments covering antigenic epitopes and synthesized a set of peptides derived from the α-fodrin protein. These recombinant proteins and synthesized peptides were subjected to screening with pSS patients sera, respectively. The peptide with the strongest immunoreactivity was used as antigenic peptide to define further the role of anti-α-fodrin-peptide antibodies in the sera of 135 patients with pSS, 48 patients with systemic lupus erythematosus (SLE), 88 patients with rheumatoid arthritis (RA) and 83 normal controls. Our data showed that the N-terminal peptide of amino acids 46–59 (N46) of α-fodrin 560 was the epitope with strongest antigenicity. The prevalences of anti-N46 peptide antibodies (α-N46PA) in patients with pSS, SLE, RA and normal controls were 78·5%, 10·4%, 21·6% and 6·0%, respectively. The sensitivity and specificity of the autoantibodies in pSS were 78·5% and 86·8%, respectively. These results suggest the α-N46PA which shows highest sensitivity and specificity is of significance to develop an effective diagnostic approach for pSS.

Monoclonal antibodies (McAb) were produced to detect semicarbazide (SEM), a metabolite as a marker residue of nitrofurazone in animal food production. A carboxyphenyl derivative (CPSEM) of SEM was synthesized following derivatisation with 4-carboxybenzaldehyde (CBA). CPSEM was purified by recrystallization and conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) as immunogen or coating antigen, respectively. Hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with CPSEM–BSA. Hybridomas 1F10 and 4F2 secreting antibodies against CPSEM were obtained and subcloned. Ascites of monoclonal antibodies were prepared by injecting 1 × 10 6 cells of hybridoma 1F10 into mice abdomen. McAb obtained from hybridoma 1F10 was highly specific for CPSEM and had no cross-reaction with various nitrofuran metabolites and a range of veterinary drugs. The sensitivity of the McAb to SEM was 0.01 ng mL −1 and the IC 50 value was 1.3 ng mL −1 (SEM in the form of CPSEM). McAb 1F10 is suitable to develop an immunoassay for SEM with sufficient sensitivity for monitoring nitrofurazone residues.

A simple and enzyme-free strip biosensor for the amplified detection of Pb(2+) has been constructed based on a catalytic DNA circuit. This assay is ultrasensitive, enabling the visual detection of Pb(2+) concentrations as low as 10 pM without instrumentation.

A lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles has been developed for the visual detection of nucleic acids with a detection limit of 0.01 fM.

A simple label-free method for the detection of Pb(2+) ions with high selectivity and sensitivity has been developed by using random double-strand DNA-templated formation of copper nanoparticles as novel fluorescence probes in aqueous solution.

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.

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.

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF(T1799A) oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF(T1799A) DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF(T1799A) DNA in complex human serum with excellent recovery (94-103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.

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+).

A direct ELISA was established for the fast detection of semicarbazide (SEM) using a novel biotin derivative. Without a tedious extraction procedure, as low as 0.07 μg L(-1) of SEM could be detected reproducibly. This assay has better recovery and accuracy than competitive ELISA.

The detection of nucleic acid is of central importance for the diagnosis of genetic diseases, infectious agents, and biowarfare agents. Traditional strategies and technologies for nucleic acid detection are time-consuming and labor-intensive. Recently, isothermal strand-displacement reaction-based lateral flow biosensors have attracted a great deal of research interest because they are sensitive, simple, fast, and easy to use. Here, we describe a lateral flow biosensor based on isothermal strand-displacement polymerase reaction and gold nanoparticles for the visual detection of nucleic acid.

A double-stranded DNA (dsDNA) mediated sandwich assay was developed for quantitative detection of transcription factors. The detection limit for human recombinant c-jun protein is 2.5 ng, and for c-jun protein the limit is as low as 0.625 μg of cell lysate.

The first example of strip logic gates ("OR" and "AND" functions) for proteins and small molecules has been constructed on the basis of target-induced self-assembly of split aptamer fragments. Using thrombin and ATP as inputs, the corresponding split/integrated aptamers as molecular recognition elements, and gold nanoparticles as a tracer, the output signals can be directly visualized by observing the red bands on the test zones of the strips. The assay is simple, easy to perform, and cost-effective, allowing portable analysis at ambient temperature. The strip logic system is resistant to nonspecific interfering agents and can operate effectively even in human serum samples. Such logic strips hold great promise for application in intelligent point-of-care and in-field diagnostics.

In this article, we describe a disposable nucleic acid biosensor (DNAB) for low-cost and sensitive detection of nucleic acid samples in 15 min. Combining the unique optical properties of gold nanoparticles (Au-NP) and the high efficiency of chromatographic separation, sandwich-type DNA hybridization reactions were realized on the lateral flow strips, which avoid multiple incubation, separation, and washing steps in the conventional nucleic acid biosensors. The captured Au-NP probes on the test zone and control zone of the biosensor produced the characteristic red bands, enabling visual detection of nucleic acid samples without instrumentation. The quantitative detection was performed by reading the intensities of the produced red bands with a portable strip reader. The parameters (e.g., the concentration of reporter probe, the size of Au-NP, the amount of Au-NP-DNA probe, lateral flow membranes, and the concentration of running buffer) that govern the sensitivity and reproducibility of the sensor were optimized. The response of the optimized device is highly linear over the range of 1-100 nM target DNA, and the limit of detection is estimated to be 0.5 nM in association with a 15 min assay time. The sensitivity of the biosensor was further enhanced by using horseradish peroxidase (HRP)-Au-NP dual labels which ensure a quite low detection limit of 50 pM. The DNAB has been applied for the detection of human genomic DNA directly with a detection limit of 2.5 microg/mL (1.25 fM) by adopting well-designed DNA probes. The new nucleic acid biosensor thus provides a rapid, sensitive, low cost, and quantitative tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and detection of infectious agents or warning against biowarfare agents.