Hemoglobin content is recognized as a momentous and fundamental physiological indicator, especially the precise detection of trace hemoglobin is of great significance for early diagnosis and prevention of tumors, cancer, organic injury, etc. Therefore, high-sensitivity hemoglobin detection is imperative. However, effective detection methods and reliable detection systems are still lacking and remain enormous challenges. Herein, we present a synthetical strategy to break through the existing bottleneck based on polarization-differential spectrophotometry and high-performance single-frequency green fiber laser. Importantly, this framework not only has precisely extracted the two-dimensional information of intensity and polarization during the interaction between laser and hemoglobin, but also has taken advantage of the high monochromaticity and fine directivity in the optimized laser source to reduce the undesirable scattered disturbance. Thus, the hemoglobin detection sensitivity of 7.2 × 10-5 g/L has advanced a hundredfold compared with conventional spectrophotometry, and the responsive dynamic range is close to six orders of magnitude. Results indicate that our technology can realize high-sensitivity detection of trace hemoglobin content, holding promising applications for precision medicine and early diagnosis as an optical direct and fast detection method.

Here we develop robust noncovalent spherical nucleic acid enzymes (SNAzymes) for direct electrochemiluminescence (ECL) detection of acute myocardial infarction (AMI) related endogenous microRNAs in both circulating blood and cardiomyocytes, which circumvents the need for time-consuming signal amplification widely used in previous counterparts. It mainly relies on the super peroxidase-like activity of the designed noncovalent SNAzymes, promoted by a few nucleotides flanking on the 3'-terminals of common parallel G-quadruplexes (G4). For this reason, an unmodified G4 with an A5T30 head is well chosen and then attached robustly onto bare AuNPs via microwave-assisted heating-drying. A probe strand is meanwhile attached onto SNAzymes, enabling the target microRNA-triggered formation of a Y-shaped junction together with a capture strand tethered to a DNA tetrahedron on the electrode surface. The utilization of this tetrahedral nanoscaffold favors the ECL readout and thereby contributes to high sensitivity of the sensing platform. In this way, an AMI-related microRNA, miR-499, can be probed in a wide linear range, with a detection limit of 33 aM and high selectivity over other analogues. Furthermore, our developed sensing platform is employed to analyze endogenous miR-499 in AMI patients' blood, revealing an apparently higher level than the mean value of the healthy. What it means to patients, heart injury, is elucidated by comparing the miR-499 levels of cardiomyocytes and other tissue cells, with endogenous miR-16 as an intrinsic reference.

Mitochondrial genetic diseases are often characterized by heteroplasmic single nucleotide polymorphisms (SNPs) where both wild-type (WT) and mutant-type (MT) coexist, making detection of accurate SNP abundance critical for diagnosis. Here, we present RatioCRISPR, an automated ratiometric biochip sensor based on the CRISPR/Cas12a system for detecting multiple heteroplasmic SNPs in mitochondrial DNA (mtDNA). The ratiometric sensor output is only influenced by the relative abundance of WT and MT, with minimal impact from sample concentration. Biochips allow the simultaneous detection of multiple SNP sites for more accurate disease diagnosis. RatioCRISPR can accurately detect 8 samples simultaneously within 25 min with a limit of detection (LOD) of 15.7 aM. We successfully detected 13 simulated samples of three mtDNA point mutations (m.3460G>A, m.11778G>A, and m.14484T>C), which lead to Leber's hereditary optic neuropathy (LHON) and set a threshold (60%) of heteroplasmy to evaluate disease risk. This automated and accurate biosensor has broad applications in diagnosing multiple SNPs, especially those with heteroplasmic variations, making it an advanced and convenient tool for mtDNA disease diagnosis.

Nanozymes with enzyme-like characteristics have drawn wide interest but the catalytic activity and substrate selectivity of nanozymes still need improvement. Herein, Se-vacancy-rich TiSe2-x@Au nanocomposites are designed and demonstrated as nanozymes. The TiSe2-x@Au nanocomposites show excellent peroxidase-like activity and the chromogenic substrate p-phenylenediamine (PPD) can be selectively oxidized to compounds that exhibit an absorption peak at 413 nm that differs from that of self-oxidation or generally oxidized species, suggesting high catalytic activity and strong substrate selectivity. Theoretical calculations reveal that the PPD adsorption geometry at Se vacancies with an adsorption energy of -3.00 eV shows a unique spatial configuration and charge distribution, thereby inhibiting the free reaction and promoting both the activity and selectivity in PPD oxidation. The TiSe2-x@Au colorimetric system exhibits a wide linear range of 0.015 mM-0.6 mM and a low detection limit of 0.0037 mM in the detection of glucose. The blood glucose detection performance for human serum samples is comparable to that of a commercial glucose meter in the hospital (relative standard deviation < 6%). Our findings demonstrate a new strategy for rapid and accurate detection of blood glucose and our results provide insights into the future design of nanozymes.

To better respond to biosecurity issues, we need to build good technology and material reserves for pathogenic microorganism screening. Here, we designed an electrochemical/optical signal probe with a common fluorophore and an electrochemically active group, breaking the previous perception that the signal probe is composed of a fluorophore and a quenching group and realizing the response of three signals: electrochemistry, fluorescence, and direct observation. Then, we proposed a homogeneous electrochemical nucleic acid detection system based on CRISPR/Cas named "HELEN-CR" by integrating free electrochemical/optical signal probes and Cas13a cleavage, achieving a limit of detection of 1 pM within 25 min. To improve the detection sensitivity, we applied recombinase polymerase amplification to amplify the target nucleic acid, achieving a limit of detection of 30 zM within 45 min. Complemented by our self-developed multi-chamber microfluidic chip and portable electrochemical instrument, simultaneous detection of multiple pathogens can be achieved within 50 min, facilitating minimally trained personnel to obtain detection results quickly in a difficult environment. This study proposes a simple, scalable, and general idea and solution for the rapid detection of pathogenic microorganisms and biosecurity monitoring.

As a unique class of dynamic nanostructures, biomimetic DNA walking machines that exhibit geometrical complexity and nanometre precision have gained great success in photoelectrochemical (PEC) bioanalysis. Despite certain achievements, the slow reaction kinetics and low processivity severely restrict the amplification efficiency of the DNA walker-mediated biosensors. Herein, by taking advantage of efficient DNA rolling machines, a three-dimensional (3D) DNA nanomachine-mediated paper-based PEC device for speedy ultrasensitive detection of miR-486-5p was successfully constructed. To achieve it, a novel In2S3/SnS2 sensitized heterojunction was firstly in-situ grown on the Au-modified paper fibers and implemented as the photoanode with effective separation of photogenerated carriers to achieve an enhanced initial photocurrent. Subsequently, the copper hexacyanoferrate(II)-modified CuO nanosphere was introduced as a multifunctional signal regulator via the competitive capture of electron donors and photon energy with the photoelectric layer for efficiently quenching the PEC signal. With the introduction of targets, the DNAzyme-driven DNA nanomachine with editable motion modes was gradually activated and it could continuously cleave the tracks DNA labeled quenching probes, finally achieving the recovery of PEC signal. As a proof of concept, the elaborated paper-based PEC device presented a wide linear range from 0.1 fM to 100 pM and a detection limit of 35 aM for miR-486-5p bioassay. This work provides an innovative insight to the exploitation of DNA nanobiotechnology and nucleic acid signal amplification strategy.

The simultaneous genotyping of multiple single nucleotide polymorphisms (SNPs) in genomic DNA derived from organisms holds significant potential for applications such as precision medicine and food product authentication. However, conventional assay technologies including qPCR-based techniques, microarrays, and hydrogel-based assays face limitations in efficient multiplexing of SNPs, particularly for large-size DNA beyond kilobase scales, due to constraints in multiplex capability, specificity, or sensitivity. In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed ∼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.

Micro/nanomotors (MNMs) emerge as a vital candidate for biosensing due to its nano-size structure, high surface-to-area ratio, directional mobility, biocompatibility, and ease of functionalization, therefore being able to detect objects with high efficiency, precision, and selectivity. The driving mode, nanostructure, materials property, preparation technique, and biosensing applications have been thoroughly discussed in publications. To promote the MNMs-based biosensors from in vitro to in vivo, it is necessary to give a comprehensive discussion from the perspective of sensing performances enhancement. However, until now, there is few reviews dedicated to the systematic discussion on the multiple performance enhancement schemes and the current challenges of MNMs-based biosensors. Bearing it in mind and based on our research experience in this field, we summarized the enhancement methods for biosensing properties such as sensitivity, selectivity, detection time, biocompatibility, simplify system operation, and environmental availability. We hope that this review provides the readers with fundamental understanding on performance enhancement schemes for MNMs-based biosensors.

Ratiometric fluorescent sensors can suppress the interference of factors unrelated to analysis due to their built-in self-calibration characteristics, which exhibit higher sensitivity and more obvious visual detection in the process of qualitative and quantitative analysis. Herein, we constructed a ratiometric fluorescence probe based on fluorescent/colorimetric dual-mode method for the determination of arginine by encapsulating rhodamine B in-situ into UiO-66-NH2 MOFs (UiO-66-NH2@RhB). The as-prepared probe showed dual-emission characteristics under a single excitation wavelength. The fluorescence intensity of UiO-66-NH2 was increased significantly by arginine, while the emission peak intensity of rhodamine B remained stable, resulting in a single-signal response with fixed reference. Furthermore, the practicality of the presented sensor was successfully validated by quantitative detection of arginine in human serum. More significantly, paper-based sensors for arginine detection were devised by using carboxymethyl cellulose modified filter papers. Under the irradiation of ultraviolet light, the paper-based sensors would produce obvious color variation from lightpink to bluish violet. This work provided a convenient and efficient method for on-site detection of arginine.

In living organisms, chiral molecules have specific chiral conformations that produce different physiological effects. Ribose is one of the components of RNA, which mainly plays a role in regulating biological activity. Inspired by the biological recognition of sugars, functional chiral surfaces for recognizing L-ribose through non-covalent interactions were constructed. In the strategy of this study, a functional chiral gold surface based on host-guest interactions was constructed through the assembly of the host molecule single-function alynyl pillar[5]arene(SAP5) and the guest molecule (S) -mandelate-violet (SMV). The association constant of SMV and SAP5 was calculated to be 2.95×104 M-1, with a binding ratio of 1:1. By impedance and contact angle detection, the constructed functional interface has good detection effect on L-ribose in the range of 1×10-7 M to1× 10-2 M. In addition, CV was disassembled from the aromatic cavity of pillar[5]arene after adding zinc powder and it can repeat five times with good recyclability, thus achieving the organic combination of interface recognition and intelligence.

Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO2 nanosponge (EDA2@hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA2@hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics.

Toehold-mediated strand displacement (TMSD) is widely employed in constructing a wide range of chemical reaction networks. In TMSD, single-stranded DNA or RNA can fold back upon itself to form a local short double-strand structure often hindering bimolecular hybridization. Here, based on series and parallel circuits, we introduce two mechanisms: series toehold-mediated strand displacement (STMSD) and parallel toehold-mediated strand displacement (PTMSD). These mechanisms can be highly effective when the target area is blocked by a secondary structure. In addition, these systems allow regulating the reaction rates spanning three to five orders of magnitude by adjusting the length of the two toeholds with the added advantage of multifunctional regulation and selectivity. To demonstrate the impressive function of this approach, a logic operation system based on STMSD was constructed to simulate the signal processing of a half-adder. We believe that the introduction of series and parallel toeholds will provide design flexibility contributing to the development of molecular computers, molecular robotics, and DNA-based biosensors.

Chronic wounds caused due to bacterial biofilms are detrimental to a patient, and an immediate diagnosis of these bacteria can aid in an effective treatment, which is still an unmet clinical need. An instant and accurate identification of bacterial type could be made by utilizing the Toll-Like Receptors (TLRs) combined with Myeloid Differentiation factor 2 (MD-2). Given this, we have developed an electrochemical sensing platform to identify the gram-negative (gram-ve) bacteria using TLR4/MD-2 complex. The nonthermal plasma (NTP) technique was utilized to functionalize amine groups onto the carbon surface to fabricate cost-effective carbon paste working electrodes (CPEs). The proposed electrochemical sensor platform with a specially engineered electrochemical cell (E-Cell) identified the Escherichia coli (E. coli) in a wide linear range of 1.5×10° - 1.5×106 C.F.U./mL, accounting for a very low detection limit of 0.087 C.F.U./mL. The novel and cost-effective sensor platform identified gram-ve bacteria predominantly in a mixture of gram positive (gram+ve) bacteria and fungi. Further, towards real-time detection of bacteria and point-of-care (PoC) applications, the effect of the pond water matrix was studied, which was minimal, and the sensor could identify E. coli concentrations selectively, showing the potential application of the proposed platform towards real-time bacterial detection.

Microalgal sensors are widely recognized for their high sensitivity, accessibility, and low cost. However, the current dilemma of motion-induced spatial phase changes and concentration-related multiple scattering interferes with induced test instability and limited sensitivity, which has hindered their practical applications. Here, a differentiated strategy, named confinement-enhanced microalgal biosensing (C-EMB), is developed and proposed to pave the way. The in-situ printed microgel trap is designed to confine Chlamydomonas reinhardtii individuals, stabilizing their spatial phase. The microgel trap arrays are introduced to eliminate the multiple scattering of microalgae, breaking the existing effective concentration in traditional microalgal sensing and enabling sensitive assays. The integration with lab-on-a-chip technology and a developed digital imaging algorithm empower portable and automated detection. With this system, a microalgae analyzer is developed for atrazine detection, featuring a linear range of 0.04-100 μg/L. We assess the system's performance through practical atrazine assays on commercial food, using a double-blind test against a standard instrument. Our results demonstrate the good accuracy and test stability of this system with the mean bias atrazine detection in corn and sugarcane juice samples (SD) were 1.661 μg/L (3.122 μg/L) and 3.144 μg/L (4.125 μg/L), respectively. This method provides a new paradigm of microalgal sensors and should advance the further applications of microalgal sensors in commercial and practical settings.

The growing need for personalized, accurate, and non-invasive diagnostic technology has resulted in significant advancements, from pushing current mechanistic limitations to innovative modality developments across various disease-related biomarkers. However, there still lacks clinical solutions for analyzing multiple biomarkers simultaneously, limiting prognosis for patients suffering with complicated diseases or comorbidities. Here, we conceived, fabricated, and validated a multifrequency impedance cytometry apparatus with novel frequency-sensitive barcoded metal oxide Janus particles (MOJPs) as cell-receptor targeting agents. These microparticles are modulated by a metal oxide semi-coating which exhibit electrical property changes in a multifrequency electric field and are functionalized to target CD11b and CD66b membrane proteins on neutrophils. A multi-modal system utilizing supervised machine learning and simultaneous high-speed video microscopy classifies immune-specific surface receptors targeted by MOJPs as they form neutrophil-MOJP conjugates, based on multivariate multifrequency electrical recordings. High precision and sensitivity were determined based on the type of MOJPs conjugated with cells (>90% accuracy between neutrophil-MOJP conjugates versus cells alone). Remarkably, the design could differentiate the number of MOJPs conjugated per cell within the same MOJP class (>80% accuracy); which also improved comparing electrical responses across different MOJP types (>75% accuracy) as well. Such trends were consistent in individual blood samples and comparing consolidated data across multiple samples, demonstrating design robustness. The configuration may further expand to include more MOJP types targeting critical biomarker receptors in one sample and increase the modality's multiplexing potential.

Doping sorted graphene quantum dots (GQDs) with heteroatoms and functionalizing them with amino acid could improve their radiative recombination and two-photon properties-including their excitation-wavelength-independent photoluminescence from the ultraviolet to the near-infrared-I (NIR-I) region, absorption, quantum yield, absolute cross section, lifetime, and radiative-to-nonradiative decay ratio-under two-photon excitation (TPE) at a low excitation energy and short photoexcitation duration, as determined using a self-made optical microscopy system with a femtosecond Ti-sapphire laser. Four types of sorted GQDs were investigated: undoped GQDs, nitrogen-doped GQDs (N-GQDs), amino-functionalized GQDs (amino-GQDs), and N-doped and amino-functionalized GQDs (amino-N-GQDs). Among them, the sorted amino-N-GQDs are effective as a two-photon photosensitizer and generate the highest quantity of reactive oxygen species for the elimination of multidrug-resistant cancer cells through two-photon photodynamic therapy (PDT). Larger amino-N-GQDs result in a greater number of C-N and N-functionalities, leading to a superior photochemical effect and more favorable intrinsic luminescence properties, making the dots effective contrast agents for tracking and localizing cancer cells during in-depth bioimaging in a three-dimensional biological environment under TPE in the NIR-II region. Overall, this study highlights the potential of large amino-N-GQDs as a material for future application to dual-modality two-photon PDT and biomedical imaging.

Microfluidic technology has emerged as a promising tool in various applications, including biosensing, disease diagnosis, and environmental monitoring. One of the notable features of microfluidic devices is their ability to selectively capture and release specific cells, biomolecules, bacteria, and particles. Compared to traditional bulk analysis instruments, microfluidic capture-and-release platforms offer several advantages, such as contactless operation, label-free detection, high accuracy, good sensitivity, and minimal reagent requirements. However, despite significant efforts dedicated to developing innovative capture mechanisms in the past, the release and recovery efficiency of trapped particles have often been overlooked. Many previous studies have focused primarily on particle capture techniques and their efficiency, disregarding the crucial role of successful particle release for subsequent analysis. In reality, the ability to effectively release trapped particles is particularly essential to ensure ongoing, high-throughput analysis. To address this gap, this review aims to highlight the importance of both capture and release mechanisms in microfluidic systems and assess their effectiveness. The methods are classified into two categories: those based on physical principles and those using biochemical approaches. Furthermore, the review offers a comprehensive summary of recent applications of microfluidic platforms specifically designed for particle capture and release. It outlines the designs and performance of these devices, highlighting their advantages and limitations in various target applications and purposes. Finally, the review concludes with discussions on the current challenges faced in the field and presents potential future directions.

Point-of-care testing (POCT) technology is urgently required owing to the prevalence of the Internet of Things and portable electronics. In light of the attractive properties of low background and high sensitivity caused by the complete separation of excitation source and detection signal, the paper-based photoelectrochemical (PEC) sensors, featured with fast in analysis, disposable and environmental-friendly have become one of the most promising strategies in POCT. Therefore, in this review, the latest advances and principal issues in the design and fabrication of portable paper-based PEC sensors for POCT are systematically discussed. Primarily, the flexible electronic devices that can be constructed by paper and the reasons why they can be used in PEC sensors are expounded. Afterwards, the photosensitive materials involved in paper-based PEC sensor and the signal amplification strategies are emphatically introduced. Subsequently, the application of paper-based PEC sensors in medical diagnosis, environmental monitoring and food safety are further discussed. Finally, the main opportunities and challenges of paper-based PEC sensing platforms for POCT are briefly summarized. It provides a distinct perspective for researchers to construct paper-based PEC sensors with portable and cost-effective, hoping to enlighten the fast development of POCT soon after, as well as benefit human society.

Pandemics as the one we are currently facing, where fast-spreading viruses present a threat to humanity, call for simple and reliable methods to perform early diagnosis, enabling detection of very low pathogen loads even before symptoms start showing in the host. So far, standard polymerase chain reaction (PCR) is the most reliable method for doing so, but it is rather slow and needs specialized reagents and trained personnel to operate it. Additionally, it is expensive and not easily accessible. Therefore, developing miniaturized and portable sensors which perform early detection of pathogens with high reliability is necessary to not only prevent the spreading of the disease but also to monitor the effectiveness of the developed vaccines and the appearance of new pathogenic variants. Thus, in this work we develop a sensitive microfluidic impedance biosensor for the direct detection of SARS-CoV-2, towards a mobile point-of-care (POC) platform. The operational parameters are optimized with the aid of design-of-experiment (DoE), for an accurate detection of the viral antigens using electrochemical impedance spectroscopy (EIS). We perform the biodetection of buffer samples spiked with fM concentration levels and validate the biosensor in a clinical context of relevance by analyzing 15 real patient samples up to a Ct value (cycle threshold) of 27. Finally, we demonstrate the versatility of the developed platform using different settings, including a small portable potentiostat, using multiple channels for self-validation, as well as with single biosensors for a smartphone-based readout. This work contributes to the rapid and reliable diagnostics of COVID-19 and can be extended to other infectious diseases, allowing the monitoring of viral load in vaccinated and unvaccinated people to anticipate a potential relapse of the disease.

Since the heart pumps out the blood through the excitation-contraction coupling, simultaneous monitoring of the electrical and mechanical characteristics is beneficial for comprehensive diagnosis of cardiac disorders. Currently, these characteristics are monitored separately with electrocardiogram (ECG) and medical imaging techniques. This work presents a fully implantable device named mechano-electrocardiogram (MECG) sensor that can measure mechanocardiogram (MCG) and ECG together. The key to the success is fabrication of permeable electrodes on a single low-modulus porous nanofiber mat, which helps immediate adhesion of the sensor on the tissue. A strain-insensitive electrode is used as the ECG electrode and a strain-sensitive electrode is used for MCG. The MECG device is implanted subcutaneously in the skin above the heart of the rat. Through a vasopressor (phenylephrine) injection test, the MECG signals indicate that the MCG amplitude is related with blood pressure and the ECG peak interval is more related with heart rate. These results confirm that the MECG device is clinically meaningful for continuous and comprehensive monitoring of the electrical and mechanical characteristics of the heart.