A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.

Sulfhydryl-functionalized 3D MXene-AuNPs enabled electrochemical sensors for the selective determination of Pb2+, Cu2+ and Hg2+ in grain.

Herein, we made 3D MXene-AuNPs by in situ growth of gold nanoparticles (AuNPs) on the surface of MXene by chemical reduction method, and then introduced three sulfhydryl (-SH) compounds as functionalized modifiers attached to the AuNPs to form a highly selective composite material for the detection of Pb2+, Cu2+, and Hg2+, respectively. The doping of AuNPs changes the microstructure of 2D MXene and generates more active sites. On a sensing platform based on ITO array electrodes, the detection system was optimised with sensitivities up to 1.157, 0.846 and 0.799 µA·µg-1Lcm-2 (Pb2+, Cu2+, and Hg2+). The selectivity of MXene@AuNPs was effectively improved by sulfhydryl group modification. In the range of 1-1300 µg L-1, the detection limits of three ions were 0.07, 0.13 and 0.21 µg L-1. In addition, this method can efficiently and accurately detect heavy metal ions in four cereal samples with consistent results with inductively coupled plasma mass spectrometry.

Fe Single-Atom Carbon Dots Nanozyme Collaborated with Nucleic Acid Exonuclease III-Driven DNA Walker Cascade Amplification Strategy for Circulating Tumor DNA Detection.

Circulating tumor DNA (ctDNA), as a next-generation tumor marker, enables early screening and monitoring of cancer through noninvasive testing. Exploring the development of new methods for ctDNA detection is an intriguing study. In this work, a unique electrochemical biosensor for the ctDNA detector was constructed in the first utilizing Fe single-atom nanozymes-carbon dots (SA Fe-CDs) as a signaling carrier in collaboration with a DNA walker cascade amplification strategy triggered by nucleic acid exonuclease III (Exo III). The electrochemical active surface area of AuNPs/rGO modified onto a glassy carbon electrode (AuNPs/rGO/GCE) was about 1.43 times that of a bare electrode (bare GCE), with good electrical conductivity alongside a high heterogeneous electron transfer rate (5.81 × 10-3 cm s-1), that is, as well as the ability to load more molecules. Sequentially, the DNA walker cascade amplification strategy driven by Exo III effectively converted the target ctDNA into an amplified biosignal, ensuring the sensitivity and specificity of ctDNA. Ultimately, the electrochemical signal was further amplified by introducing SA Fe-CDs nanozymes, which could serve as catalysts for 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with facile responding (Vmax = 0.854 × 10-6 M s-1) and robust annexation (Km = 0.0069 mM). The integration of the triple signal amplification approach achieved detection limits as low as 1.26 aM (S/N = 3) for a linearity spanning from 5 aM to 50 nM. In this regard, our proposal for a biosensor with exceptional assay properties in complicated serum environments had great potential for early and timely diagnosis of cancer.

A wearable flexible electrochemical biosensor with CuNi-MOF@rGO modification for simultaneous detection of uric acid and dopamine in sweat.

BACKGROUND: In health assessment and personalized medical services, accurate detection of biological markers such as dopamine (DA) and uric acid (UA) in sweat is crucial for providing valuable physiological information. However, there are challenges in detecting sweat biomarkers due to their low concentrations, variations in sweat yield among individuals, and the need for efficient sweat collection. RESULTS: We synthesized CuNi-MOF@rGO as a high-activity electrocatalyst and investigated its feasibility and electrochemical mechanism for simultaneously detecting low-concentration biomarkers UA and DA. Interaction between the non-coordinating carboxylate group and the sample produces effective separation signals for DA and UA. The wearable biomimetic biosensor has a wide linear range of 1-500 µM, with a detection limit of 9.41 µM and sensitivity of 0.019 µA µM-1 cm-2 for DA, and 10-1000 µM, with a detection limit of 9.09 µM and sensitivity of 0.026 µA µM-1 cm-2 for UA. Thus, our sensor performs excellently in detecting low-concentration biomarkers. To improve sweat collection, we designed a microfluidic-controlled device with hydrophilic modification in the microchannel. Experimental results show optimal ink flow at 2% concentration. Overall, we developed an innovative and highly active electrocatalyst, successfully enabling simultaneous detection of low-concentration biomarkers UA and DA. SIGNIFICANCE: This study provides a strategy for sweat analysis and health monitoring. Moreover, the sensor also showed good performance in detecting real sweat samples. This study has shown great potential in future advances in sweat analysis and health monitoring.

Label-free fluorescent sensor for sensitive detection of ctDNA based on water stabilized CsPbBr3 nanosheet.

The detection of circulating tumor DNA (ctDNA), as a practical liquid biopsy technique, was of great significance for the study of cancer diagnosis and prognosis. However, reported methods for detection ctDNA still have some limitations, such as tedious process and high cost. In this study, CsPbBr3 nanosheet (CsPbBr3 NS) with high water stability was prepared by etching, and its fluorescence intensity could be stably stored for 1 year. The Ti3C2Tx possessed high quenching efficiency for CsPbBr3 NS and the HOMO-LUMO orbital study revealed that the PET mechanism was responsible for fluorescence quenching. And the Ti3C2Tx showed stronger affinity towards single-stranded DNA (ssDNA), as compared with double-stranded DNA (dsDNA). The probe ssDNA could be adsorbed on the surface of Ti3C2Tx through π-π stacking. After the targets were recognized by probe ssDNA to form dsDNA, its affinity with Ti3C2Tx decreased and the active site of Ti3C2Tx recovered, causing a high quenching efficiency on CsPbBr3 NS. Based on this, a label-free fluorescent biosensor was designed for the sensitive detection of ctDNA (EGFR 19 Dels for non-small cell lung cancer, NSCLC). Under the optimal experimental conditions, this biosensor exhibited a detection limit of 180 fM and a linear range of 50 pM-350 pM with amplification of magnetic beads through strand displacement reaction. In addition, this sensor was applied to the detection of ctDNA in serum samples and cells lysates. This method for ctDNA detection was expected to have great potential for biomarker detection in the field of liquid biopsy.

Pattern-based colorimetric sensor array chip for discrimination of Baijiu aromas.

Gas mixtures are comprised of numerous complex components, making the accurate identification a continuing challenge due to the significant limitations of existing detection methods. Herein, we developed a low-cost and sensitive pattern-based colorimetric sensor array chip for the identification of typical gas mixtures - Baijiu aroma. Specifically, three nanomaterials (AuNPs, MoS2 and ZIF-8) were prepared to adsorb gas molecules and enhance the reaction of trace gases with sensor arrays. The colorimetric sensor array chip took only 5 min to complete the recognition of Baijiu aromas and effectively avoided recognition errors caused by sommelier olfactory fatigue. Notably, the hierarchical cluster analysis (HCA) revealed no confusion or errors in the results of 80 tests across the five trials involving 16 commercial Baijius. Even fake Baijius with similar ingredients could be easily identified, demonstrating the excellent analytical capabilities of the system in Baijiu identification and its significant potential for quality control of Baijius.

A three-modal fluorescent sensor harnessing diverse luminescent mechanisms for the purpose of segmented Baijiu identification.

The classification and verification of segmented Baijiu hold significant importance as they profoundly influence the blending and overall quality of the Baijiu. Our scholarly investigation yielded a fluorescent sensor with three luminescent modes by integrating Tb3+ and RHB into UiO-66. The interplay between carboxyl-containing compounds and RHB/Tb@TLU-2 orchestrates a harmonious molecular association, where the convergence of carboxyl groups with Tb3+ yields a resonating impact on the antenna effect of BDC-SO3-. Furthermore, the acidity and alkalinity of reactants induced a charge transfer interaction between BDC-NH2 and Zr4+ and led to structural changes in RHB/Tb@TLU-2, resulting in observable fluorescence signal variations across the three emission centers. The sensor array successfully identified eight organic acids, achieving an impressive 97.5 % accuracy in discerning segmented Baijiu samples from four Baijiu pits. This meticulous methodology prioritizes simplicity, swiftness, and effectiveness, paving the path for comprehensive segmented Baijiu analysis in the esteemed realm of Brewing production.

Wearable electrochemical patch based on iron nano-catalysts incorporated laser-induced graphene for sweat metabolites detection.

The development of wearable devices shows great application potential in health management. In this work, we propose the fabrication of a novel wearable electrochemical patch and prove its application in sweat metabolites detection. The patch is developed based on iron nano-catalysts incorporated laser-induced graphene (FeNCs/LIG), which is a newly integrated sensing electrode with unique three-dimensional nanostructure and good electrocatalytic activity. It shows desirable sensing performances for sweat metabolites including tyrosine (Tyr) and uric acid (UA) molecules. The detection limit of Tyr and UA can reach 5.11 µM and 1.37 µM, respectively. Besides, density functional theory calculation deeply reveals that the Fe active sites of FeNCs play an important role in molecule adsorption and electron transference, thus promoting sensing performance. To realize wearable application, a dual-channel hydrogel chip is designed and assembled with FeNCs/LIG. The developed patch is successfully utilized to accurately determination of Tyr and UA in sweat. This work is expected to provide a new non-invasive strategy for evaluating amino acid intake and metabolic level.

Ultra-sensitive electrochemical sensors through self-assembled MOF composites for the simultaneous detection of multiple heavy metal ions in food samples.

Using an assemble-able MOF material, we successfully constructed an ultra-sensitive electrochemical sensor based on Bi2CuO4@Al-MOF@UiO-67 nanocomposite material, in order to investigate the adsorption properties of the Bi2CuO4@Al-MOF@UiO-67 functional material on the heavy metal ion. The Cd2+, Cu2+, Pb2+ and Hg2+ can be detected at the same time. Selective recognition and enrichment of various metal ions on different substrates can be achieved through the assembly of a large number of Al-MOF and UiO-67-MOF nanomaterial composites with small particle sizes on the Bi2CuO4 surface. Based on this, a new type of sensor is researched and prepared, which has been shown to have good stability and reproducibility. Due to its unique assembly structure, large active surface area, excellent adsorption capacity, and high electrical conductivity, Bi2CuO4@Al-MOF@UiO-67 presents outstanding performance. In addition, the sensor also exhibits excellent electrocatalytic redox capacity and high selectivity. The adsorption capacity of Cd2+, Cu2+, Pb2+ and Hg2+ is also significantly improved under the action of the sensor electrode, however, this is not the case. The limits of detection for Cd2+, Cu2+, Pb2+ and Hg2+ were found to be 0.02 pM, 0.032 pM, 0.018 pM and 0.041 pM, respectively. In order to investigate the detection mechanism of Cd2+, Cu2+, Pb2+ and Hg2+ was adsorption properties as well as electrochemical accumulation of Bi2CuO4@Al-MOF@UiO-67 on the metal atoms were investigated. This method has been successfully applied to samples of rice, sorghum, maize, milk, honey, and tea, and has enabled the simultaneous detection of Cd2+, Cu2+, Pb2+ and Hg2+, which is of significant practical value.

A novel colorimetric and fluorometric dual-signal identification of organics and Baijiu based on nanozymes with peroxidase-like activity.

Nanozymes were nanomaterials with enzymatic properties. They had diverse functions, adjustable catalytic activity, high stability, and easy large-scale production, attracting interest in biosensing. However, nanozymes were scarcely applied in Baijiu identification. Herein, a colorimetric and fluorometric dual-signal determination mediated by a nanozyme-H2O2-TMB system was developed for the first time to identify organics and Baijiu. Since the diverse peroxidase-like activity of nanozymes, resulted in different degrees of oxidized TMB. Based on this, 21 organics were identified qualitatively and quantitatively by colorimetric method with a rapid response (<12 min), broad linearity (0.0005-35 mM), and low detection limits (a minimum of 30 nM for glutaric acids). Furthermore, the fluorometric method exhibited excellent potential for accurate determination of organics, with detection ranges of 2-200 µmol/L (LOD: 0.22 µmol/L) for l-ascorbic acid and 2-300 µmol/L (LOD: 0.59 µmol/L) for guaiacol. Finally, the sensor was successfully applied to identify fake Baijiu and Baijiu from 16 different brands.

Microswimmer-Assisted Dual-Signal Sensor for Multiple Targets in Whole Blood.

Accurate detection of biomarkers in whole blood is an important aspect of diagnostic testing but remains a challenge due to various interferences. However, using a self-calibrating two-signal strategy offers a solution that can overcome interference caused by experimental and environmental factors. Here, we proposed a novel microswimmer {methylene blue (MB)@ZIF-90@aptamer-HER2/3,3',5,5'-tetramethylbenzidine (TMB)@ZIF-90@aptamer-ER}-dual-signal (electrochemical and fluorescence) homogeneous sensor based on functionalized ZIF nanomaterials for one-step simultaneous detection of human epidermal growth factor receptor-2 (HER2) and estrogen receptor (ER) in whole blood. The proposed one-step ZIF-90 synthesis encapsulates TMB and MB with dual-signal properties. HER2 and ER aptamers adsorbed on MB@ZIF-90/TMB@ZIF-90 function as the gate switches. The microswimmer targets the HER2 and ER with adenosine triphosphate (ATP)-driven motion. When targets are present, aptamers dissociate and reduce the microswimmer's surface negative charge. The microswimmer undergoes attack and decomposition by swimming ATP due to the strong coordination force between ATP and Zn2+, leading to the release of MB and TMB. The negative charges on the surface of indium tin oxide enrich MB and TMB with positive charges, thereby increasing the intensities of electrochemical and fluorescence signals. The detection process was completed within 40 min, and the detection limits for ER and HER2 were 8.1 and 5.7 fg/mL respectively, with a linear range of 0.25-20 pg/mL.

Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays.

Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the "TTTN". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA not only achieved efficient amplification but also converted dsDNA to single-stranded DNA (ssDNA) without complicated steps. The ssDNA products activated the trans-cleavage activity of Cas12a, outputting signals. The application of Asy-RPA completely freed Cas12a from the PAM, which can be more widely used in nucleic acid detection, such as lumpy skin disease virus, with an actual detection limit as low as 1.21 × 101 copies·µL-1. More importantly, Cas12a was intolerant to mutations on ssDNA. This provided technical support for the detection and identification of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples. The detection and availability of different treatment options for treatment-resistant and WT-TB were significant for the elimination of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a was suitable for the detection of various viruses and bacteria and was a boon for the detection of dsDNA without recognizable PAM.

Field-friendly and ultra-fast detection platform without nucleic acid extraction for virus detection.

The polymeric chain reaction (PCR) has come under fire for being time-consuming, requiring expensive equipments, and requiring the extraction and purification of nucleic acids. Here, an ultra-fast and sensitive detection platform without nucleic acid extraction solved the above problems. Firstly, the RoomTemp Sample Lysis Kit released the nucleic acid in 3 min and removed the inhibition to facilitate the amplification reaction. What's more, ultra-fast PCR (UF-PCR) can complete 40 cycles in just 15 min and 50 s. To improve the sensitivity and provide more convenient reading modes, CRISPR/Cas12a was mediated to detect Lumpy skin disease virus (LSDV). The platform output fluorescence and Lateral flow dipstick (LFD) signals. The actual detection limit was 2 × 101 copies·µL-1. The portable platform realized visualization, excellent sensitivity and quick speed. In summary, the field-friendly testing platform had great potential in practical testing.

Identification of Chinese baijiu from the same brand based on a graphene quantum dots fluorescence sensing array.

The identification of Chinese baijiu is crucial to regulating the international market and maintaining legitimate rights, as the popularity, influence and awareness of baijiu are growing. A graphene quantum dot (GQD) based fluorescence sensor array is designed in this paper. Upon using only GQDs as a single sensing element, combining three different solvents improves the sensing array's detection sensitivity while simplifying material preparation and experimental detection. Adding organic substances creates intermolecular forces between the GQDs and the solvent, causing the fluorescence intensity to change. The sensor array was able to distinguish 21 types of organic matter, different ratios of quaternary mixed organic materials and 17 types of baijiu of the same brand. It also showed excellent performance in the detection of species in blind samples, with the machine learning algorithm successfully distinguishing baijiu from five other distilled spirits. The experiment provides guidance for the practical application of GQDs and provides a simple but effective reference for sensor arrays to detect baijiu.

A PVDF-based colorimetric sensor array for noninvasive detection of multiple disease-related volatile organic compounds.

Detection of human-generated volatile organic compounds (VOCs) is a new pathway for assessing health. Herein, a polyvinylidene fluoride (PVDF)-based colorimetric sensor array was designed for detecting disease-related VOCs (DVOCs) within 15 min, using a complex of Cu metal-organic framework, graphene aerogel, and dyes as response materials. Fingermaps derived from 28 DVOCs were obtained for further data processing. Pattern recognition was successfully employed in the correct discrimination of 28 DVOCs in low (10 µM), medium (100 µM), and high (300 µM) concentrations. Importantly, the sensor array also presented excellent discrimination ability and application potential when detecting VOCs produced by human cancer and normal cells. In general, VOC acquisition is noninvasive and harmless, and the PVDF-based sensor arrays are simple and visual. Such advantages expand their further application potential.

CRISPR/Cas12a and primer-assisted rolling circle amplification integrated ultra-sensitive dual-signal sensing platform for EGFR 19 detection.

Herein, we integrated CRISPR/Cas12a with primer-assisted rolling circle amplification (PARCA) to specifically detect EGFR 19 from the genome. We fused the method into fluorescent and electrochemical detection systems forming a stable and sensitive dual-signal sensing platform. The fluorescent detection system stably detected EGFR 19 in a linear range from 500 fM to 10 nM with an ultra-low background signal. The electrochemical detection system possessed a detection limit as low as 42 aM due to the introduction of nanomaterial UIO-66-NH2. The dual-signal sensing platform showed superior performance in complex serum samples and real cell genomes and provided a flexible and dynamic approach for the ultra-sensitive detection of EGFR 19.

Target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a for simultaneous detection of HPV16 and HPV18.

Simultaneous detection of multiple targets can provide important data support for clinical diagnosis and treatment. Here, we report a facile isothermal assay based on target-mediated rolling circle transcription coupling with CRISPR/Cas12a-Cas13a (TM-RCT/Cas12a-Cas13a). Through facile one-step amplification (TM-RCT), two target DNAs are converted to RNA amplified products. The simultaneous detection of HPV16 and HPV18 is then achieved by combining two CRISPR/Cas systems. This system shows excellent sensing performance and provides a universal method for simultaneous detection.

Fe Single-Atom Nanozyme-Modified Wearable Hydrogel Patch for Precise Analysis of Uric Acid at Rest.

Resting sweat analysis could provide unique insight into the metabolic levels of physiological and pathological states. However, the low secretion rate of resting sweat and the low concentration of metabolic molecules pose challenges for the development of noninvasive wearable sensors. Here, we demonstrated a wearable patch for the precise analysis of uric acid at rest. Fe single-atom nanozymes (FeSAs) with excellent electrocatalytic activity were used to develop a sensor for selective catalysis of uric acid (UA, 1-425 µM), and the catalytic mechanism of UA was later explored by density functional theory. In addition, polyaniline was integrated into the wearable patch for pH detection; thus, accurate analysis of sweat UA molecules can be achieved by pH calibration. Then, we explored the possibility of collecting resting sweat with different ratios of agarose hydrogels to reduce the sweat accumulation time. Finally, the possibility of a wearable patch for accurate UA detection in volunteer sweat samples was experimentally verified. We believe that our work provides novel insights and ideas for the analysis of resting sweat using wearable devices, further driving advancements in the field of personalized medicine.

Synthesis of N,Si co-doped carbon dots to establish a fluorescent sensor for Hg(II) detection with triple signal output.

Mercury is a heavy metal with extreme toxicity. Thus, it is of significance to develop an effective method for mercury ion detection with high performance. In this study, carbon dots doped with nitrogen and silicon (N,Si/CQDs) were successfully prepared from folic acid and N-[3-(trimethoxysily)propyl]-ethylenediamine. The N,Si/CQDs show an obvious cyan fluorescence of 460 nm with the radiation of 350 nm. The existence of mercury ions induces the fluorescence quenching of N,Si/CQDs due to photoinduced electron transfer, which was applied for the sensitive sensing of Hg(II). More importantly, the practical application of the N,Si/CQD probe was confirmed by measurements of Hg(II) in real samples of lake water, sorghum and rice. In addition, the N,Si/CQD nanoprobe was integrated on a sensing strip for specific detection of Hg(II). Quantitative measurement of Hg(II) was realized by the outstanding linearity between the diameter (or fluorescence intensity) of the fluorescence quenching ring and the concentration of mercury ions. The sensor shows potential for rapid detection with a triple signal readout on-site and represents a larger step towards practical applications.