Amplification-free Detection Research Articles

M13mp18 nanoscaffold-based strip sensor for detecting influenza A virus (H1N1)

We report the development of an amplification-free nucleic acid assay using lateral flow test strips. M13mp18 nanoscaffold signal amplification permitted the visual detection of H1N1 RNA in 20 min. M13mp18 nanoscaffold supported a set of backbone, variable, detection, and modified oligonucleotides. A bridging-type FAM probe/H1N1 RNA/M13mp18 was used in the lateral flow test strip format. The bridging-type complexes were captured on the test zone. The lower limit of detection of specific H1N1 RNA was 1 pM. The method directly detected H1N1 RNA without amplification, eliminating the risk of amplicon contamination and lessening the chance of false positives. This biosensor has potential value for field testing in resource-limited settings and monitoring viral loads in recovering patients. The amplification-free lateral flow detection of RNA provides an alternative between amplification-based RNA assays and protein antigen assays for facilitating the rapid identification of H1N1 virus during point-of-care testing.

An Amplification-Free Digital Assay Based on Primer Exchange Reaction-Mediated Botryoidal-Like Fluorescent Polystyrene Dots to Detect Multiple Pathogenic Bacteria.

Multiple and ultrasensitive detection of pathogenic bacteria is critical but remains a challenge. Here, we introduce a digital assay for multiplexed and target DNA amplification-free detection of pathogenic bacteria using botryoidal-like fluorescent polystyrene dots (PS-dots), which were first prepared through the hybridization reaction between primer exchange reaction chains and polystyrene nanospheres that encapsulated polymer dots for signal preamplification. The pathogenic bacteria's DNA was cleavaged by the argonaute (Ago) protein-mediated multiple and precise cleavage reactions, where the obtained target sequences bridged the magnetic beads (MBs) and botryoidal-like PS-dots via a hybridization reaction, and the fluorescent MB-botryoidal PS-dot complexes were utilized as digital probes based on colors and sizes for digital encoding. An artificial-intelligence-fluorescent microsphere counting algorithm was applied to identify and count the fluorescent MBs for digital readout. This digital assay combined the ultrabright botryoidal-like PS-dots with Clostridium butyricum Ago's precise enzyme cleavage properties, achieving simultaneous detection of three pathogenic bacteria with a linearity range from 102 to 106 CFU/mL without target DNA amplification within 1.5 h. This digital assay has also been applied to detect aquatic and clinical samples with accepted accuracy (98%), which offers an avenue for a next-generation multiplexed digital platform for pathogenic bacteria analysis.

Glycan-Evocated Metallization for Amplification-Free Electrochemical Detection of Glycoproteins at Low Concentration Levels.

Glycoproteins have become the most often screened tumor markers in the in vitro diagnostics. Although a large number of electrochemical methods have been proposed to sensitively detect glycoproteins, most of them involve the aid of laborious signal amplification. Herein, we report the use of glycan-evocated metallization (GlyMetal) for the amplification-free electrochemical detection of glycoproteins at low concentration levels. Briefly, the glycoproteins of interest are captured by an aptamer recognition layer, and then the glycans of targets are oxidized by NaIO4 to convert the 1,2-diol sites into aldehyde groups for the silver deposition-based metallization, followed by the electrochemical stripping assay of the deposited metallic silver for glycoprotein quantification via the established solid-state Ag/AgCl voltammetric process. As GlyMetal can enable the deposition of a large amount of metallic silver and a high signal-to-background ratio can be obtained for the solid-state Ag/AgCl voltammetric stripping assay, the developed GlyMetal-based electrochemical method is applicable to the amplification-free detection of glycoproteins. As a proof of concept, a detection limit of 1.65 pg/mL has been achieved for carcinoembryonic antigen (CEA) detection. In addition to the high selectivity, desirable results have been obtained with respect to the use of the method for CEA detection in serum samples. In consideration of the desirable simplicity, short assay time, and cost-effectiveness of the amplification-free approach, the GlyMetal-based electrochemical method shows great promise in the point-of-care detection of glycoproteins.

Amplification-free detection of Ascochyta blight in chickpea using a simple molecular beacon assay

Ascochyta blight is a major biotic stress that limits chickpea production globally. Fungicide application remains one of the effective control measures for the endemic spread. Due to the serious threat that synthetic fungicides pose to crop quality, early diagnosis of the pathogen is imperative. Whilst there have previously been several conventional lab-based diagnostic methods developed for early detection of Ascochyta rabiei, they require long assay times, specialised equipment and facilities, and trained personnel to process the samples. To overcome this challenge, a rapid amplification-free detection assay using a molecular beacon probe was developed. The method consists of a simple assembly assay that accurately detects pathogen within 30 min. The developed assay is species-specific and has a similar sensitivity level as conventional amplification-based methods. Although it is still a lab-based technique, considering the simplicity of the assay, it has a great potential to be developed further as a reliable in-field diagnostic device for early detection and quantification of fungal pathogen spores.

Nucleic Acid-Based Biological Nanopore Sensing Strategies for Tumor Marker Detection.

Cancer, which is characterized by high mortality rates, poses a significant threat to global human health. Early diagnosis is of paramount importance in managing cancer, and tumor markers have emerged as crucial indicators for achieving this goal. The advent of precision medicine has further emphasized the need for the effective detection of these markers. However, traditional detection methods are hampered by numerous limitations. In recent years, nanopore technology has emerged as a promising alternative, due to its unique physical and chemical properties, which facilitate rapid, label-free, and amplification-free detection. This Review focuses on the direct detection of tumor markers through nucleic acid analysis and indirect detection mediated by nucleic acids and facilitated by biological nanopores. Furthermore, it also discusses the challenges and prospects of applying biological nanopore sensing technology in early cancer diagnosis, underscoring its potential to revolutionize tumor marker detection.

241 Proof-of-Principle: CRISPR-Based Rapid, Amplification-Free Bacterial RNA Detection for POC Bacteremia Detection

241 Proof-of-Principle: CRISPR-Based Rapid, Amplification-Free Bacterial RNA Detection for POC Bacteremia Detection

Target-mediated silver deposition-based electrochemical biosensor for highly sensitive detection of human chorionic gonadotropin

As a glycoprotein hormone, human chorionic gonadotropin (hCG) is an established marker for pregnancy test. On the basis of the target-mediated silver deposition (TSD), in this work, we report the development of an amplification-free electrochemical biosensor for the highly sensitive detection of hCG. The detection of hCG involves the use of the affinity peptide-modified electrode for hCG capture (the CGGSSPPLRINRHILTR peptide containing the hCG-binding domain of the PPLRINRHILTR sequence is used as the affinity peptide), the oxidation of the diol sites of the glycan chains on hCG hormones into aldehyde groups by NaIO4, and the deposition of silver nanoparticles (AgNPs) for the solid-state voltammetric stripping analysis. Due to the deposition of multiple AgNPs while the solid-state Ag/AgCl voltammetric process has a high signal-to-noise ratio, the TSD-based electrochemical biosensor can be applied to the highly sensitive detection of hCG without the need for signal amplification. Under optimal conditions, the stripping current increased linearly with an increasing hCG concentration over the range from 1.0 to 25 mIU/mL, with a detection limit of 0.45 mIU/mL. Owing to the high specificity of the hCG-binding peptide PPLRINRHILTR, this electrochemical hCG biosensor exhibits high selectivity. The results of the quantitative assay of hCG in urine samples at the concentrations of 25, 10, and 1.0 mIU/mL are desirable, indicating the good anti-interference capability. As the TSD-based electrochemical biosensor allows the amplification-free detection of low-abundance hCG, it is easy to use and cost-effective, showing great promise in point-of-care assay of hCG for pregnancy test.

Waveguide-Enhanced Nanoplasmonic Biosensor for Ultrasensitive and Rapid DNA Detection.

DNA is fundamental for storing and transmitting genetic information. Analyzing DNA or RNA base sequences enables the identification of genetic disorders, monitoring gene expression, and detecting pathogens. Traditional detection techniques like polymerase chain reaction (PCR) and next-generation sequencing (NGS) have limitations, including complexity, high cost, and the need for advanced computational skills. Therefore, there is a significant demand for enzyme-free and amplification-free strategies for rapid, low-cost, and sensitive DNA detection. DNA biosensors, especially those utilizing plasmonic nanomaterials, offer a promising solution. This study introduces a novel DNA-functionalized waveguide-enhanced nanoplasmonic optofluidic biosensor using a nanogold-linked sorbent assay for enzyme-free and amplification-free DNA detection. Integrating plasmonic gold nanoparticles (AuNPs) with a glass planar waveguide (WG) and a microfluidic channel, fabricated through cost-effective, vacuum-free methods, the biosensor achieves specific detection of complementary target DNA sequences. Utilizing a sandwich architecture, AuNPs labeled with detection DNA probes enhance sensitivity by altering evanescent wave distribution and inducing plasmon resonance modes. The biosensor demonstrated exceptional performance in DNA detection, achieving a limit of detection (LOD) of 33.1 fg/mL (4.36 fM) with a rapid response time of approximately 8 min. This ultrasensitive, rapid, and cost-effective biosensor exhibits minimal background nonspecific adsorption, making it highly suitable for clinical applications and early disease diagnosis. The innovative design and fabrication processes offer significant advantages for mass production, presenting a viable tool for precise disease diagnostics and improved clinical outcomes.

Magnetic Fe3O4@MOF@aptamer-mediated entropy-driven fluorescence biosensor for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria

Rapid and early identification of pathogenic bacteria in food and clinical samples is critical for infectious disease control and precision medicine. In this study, we present a novel Magnetic Fe3O4@metal–organic framework (MOF)@Aptamer-based Entropy-driven fluorescence biosensor combined with mesophilic Clostridium butyricumArgonaute (CbAgo) for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria, termed the MAEA biosensor. In this study, magnetic zirconium-based MOF nanoparticles were modified with a phosphorylated aptamer as an efficient signal converter, which recognized the pathogens and released the complementary DNA to activate the entropy-driven circuit reaction. The highly efficient catalytic DNA circuit and isothermal enzyme-free format produce large amounts of guide DNA. CbAgo exerts precise and highly efficient endonuclease activity under the guidance of guide DNA, which makes the MAEA biosensor useful for multiplexed and DNA extraction- and amplification-free detection of pathogenic bacteria with high sensitivity (<102 CFU/mL). This study demonstrates a highly selective and reliable magnetic Fe3O4@MOF@aptamer-based biosensor using CbAgo for simultaneously detecting multiple pathogenic bacteria, providing a potential tool for other infectious disease pathogens with convenient operation and high sensitivity.

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Quantum-Dot-Encoded Beads-Enhanced CRISPR/Cas-Based Lateral-Flow Assay for the Amplification-Free, Sensitive, and Rapid Detection of Nucleic Acids in Breast Cancer.

Nucleic acid detection plays a pivotal role in the accurate diagnosis of diseases. The CRISPR/Cas detection system, noted for its significant utility in a variety of applications, often necessitates enhanced sensitivity or specific signal amplification strategies, particularly for detecting low-abundance biomarkers. In this study, we present a quantum-dot-encoded beads (QDB)-energized CRISPR/Cas12-based lateral-flow assay (QDB-CRISPR-LFA). This method enables amplification-free, sensitive, and rapid detection (<40 min) of BRCA-1. We validated our method using contrived reference samples and nucleic acids extracted from tumor cells. The QDB-CRISPR-LFA provides a visual, more rapid alternative to the traditional BRCA-1 real-time RT-PCR assay. Significantly, through the integration of CRISPR's specificity and the high signal output of QDB, the detection threshold for BRCA-1 has been reduced to the femtomolar level, representing an enhancement of 2-4 orders of magnitude over existing CRISPR/Cas detection methods. This advancement underscores the potential of our approach in advancing nucleic acid detection techniques, which is crucial for the early and precise diagnosis of diseases.

Electrokinetics with Surface-Enhanced Raman Spectroscopy for Bacterial Identification in Wastewater

Bacterial presence in wastewater serves as a crucial bioindicator for monitoring population-level infections and offering early disease outbreak warnings [1,2]. However, identifying bacteria in wastewater presents outstanding challenges; notably, current culturing or fluorescence-based methods [3] to identify bacteria are slow and expensive, unsuitable for high-throughput screening of diverse bacterial species, and may not work well in the complex wastewater matrix. Here, we harness the potential of surface chemistry and electrokinetics to introduce an innovative electro-optical approach that has the promise to detect a broad spectrum of pathogenic bacteria in wastewater.We combine surface-enhanced Raman spectroscopy (SERS) [4] with alternating current electric fields, complemented by machine learning models [5], to enable rapid and amplification-free bacteria detection in filtered wastewater, at cell concentrations as low as 104 cells/mL. First, we synthesize gold nanorods that can electrostatically bind to bacteria surfaces, allowing for SERS from cell surfaces. We collect SERS from bacteria spiked into filtered wastewater, including Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, spanning concentrations from 109 cells/mL to 104 cells/mL. Spectral clustering analysis shows that bacteria signals become more indistinguishable from wastewater as the concentrations decrease. Further, we incorporate electrokinetic effects into SERS by employing gold microelectrodes to apply electric fields, utilizing dielectrophoresis [6] to rapidly displace and concentrate bacteria based on their dielectric responses within minutes. The enrichment of bacteria with nanorods is directly visualized through microscopy, resulting in Raman signal intensities increased by up to tenfold under electrical fields for bacterial concentrations ranging from 106 down to 104 cells/mL. Such enhancement may enable the detection sensitivity to reach environmentally-relevant concentrations. Moreover, employing data science analysis, we identify biologically-relevant “fingerprint” Raman peaks characterizing proteins, nucleic acids, and lipids from bacteria surfaces, allowing for rapid identification of bacteria species in wastewater. This novel method exhibits potential for generalized pathogen detection and molecular recognition in complex liquid samples, such as wastewater, blood, and seawater.[1] Hellmér, et al. Applied and environmental microbiology. (2014).[2] Keshaviah, et al. The Lancet Global Health. (2023).[3] Jahn, et al. Nature Microbiology. (2022).[4] Tadesse, et al., Nano Lett. (2020).[5] Ho, et al., Nat. Comm. (2019).[6] Pethig. John Wiley & Sons (2010).

Peptide nucleic acid probe-assisted paper-based electrochemical biosensor for multiplexed detection of respiratory viruses

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43–25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases.

Modular Fabrication of Microfluidic Graphene FET for Nucleic Acids Biosensing.

Graphene field-effect transistors (GFETs) are widely used in biosensing due to their excellent properties in biomolecular signal amplification, exhibiting great potential for high-sensitivity and point-of-care testing in clinical diagnosis. However, difficulties in complicated fabrication steps are the main limitations for the further studies and applications of GFETs. In this study, a modular fabrication technique is introduced to construct microfluidic GFET biosensors within 3 independent steps. The low-melting metal electrodes and intricate flow channels are incorporated to maintain the structural integrity of graphene and facilitate subsequent sensing operations. The as-fabricated GFET biosensor demonstrates excellent long-term stability, and performs effectively in various ion environments. It also exhibits high sensitivity and selectivity for detecting single-stranded nucleic acids at a 10fm concentration. Furthermore, when combined with the CRISPR/Cas12a system, it facilitates amplification-free and rapid detection of nucleic acids at a concentration of 1fm. Thus, it is believed that this modular-fabricated microfluidic GFET may shed light on further development of FET-based biosensors in various applications.

Rapid and Amplification-free Nucleic Acid Detection with DNA Substrate-Mediated Autocatalysis of CRISPR/Cas12a.

To enable rapid and accurate point-of-care DNA detection, we have developed a single-step, amplification-free nucleic acid detection platform, a DNA substrate-mediated autocatalysis of CRISPR/Cas12a (DSAC). DSAC makes use of the trans-cleavage activity of Cas12a and target template-activated DNA substrate for dual signal amplifications. DSAC employs two distinct DNA substrate types: one that enhances signal amplification and the other that negatively modulates fluorescent signals. The positive inducer utilizes nicked- or loop-based DNA substrates to activate CRISPR/Cas12a, initiating trans-cleavage activity in a positive feedback loop, ultimately amplifying the fluorescent signals. The negative modulator, which involves competitor-based DNA substrates, competes with the probes for trans-cleaving, resulting in a signal decline in the presence of target DNA. These DNA substrate-based DSAC systems were adapted to fluorescence-based and paper-based lateral flow strip detection platforms. Our DSAC system accurately detected African swine fever virus (ASFV) in swine's blood samples at femtomolar sensitivity within 20 min. In contrast to the existing amplification-free CRISPR/Dx platforms, DSAC offers a cost-effective and straightforward detection method, requiring only the addition of a rationally designed DNA oligonucleotide. Notably, a common ASFV sequence-encoded DNA substrate can be directly applied to detect human nucleic acids through a dual crRNA targeting system. Consequently, our single-step DSAC system presents an alternative point-of-care diagnostic tool for the sensitive, accurate, and timely diagnosis of viral infections with potential applicability to human disease detection.

Label-free electrochemical aptasensor for the detection of HepG2 hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common liver malignancy and is characterized by increasing incidence and high mortality rates. Current methods for the screening and diagnosis of HCC exhibit inherent limitations, highlighting the ever-growing need for the development of new methods for the early diagnosis of HCC. The aim of this work was to develop a novel electrochemical aptasensor for the detection of HepG2 cells, a type of circulating tumor cells that can be used as biomarkers for the early detection of HCC. A carbon screen-printed electrode was functionalized with a composite suspension containing graphene oxide, chitosan, and polyaniline nanoparticles to increase the electrode surface and provide anchoring sites for the HepG2 cell-specific aptamer. The aptamer was immobilized on the surface of the functionalized electrode using multipulse amperometry, an innovative technique that significantly reduces the time required for aptamer immobilization. The innovative platform was successfully employed for the first time for the amplification-free detection of HepG2 cells in a linear range from 10 to 200,000 cells/mL, with a limit of detection of 10 cells/mL. The platform demonstrated high selectivity and stability and was successfully used for the detection of HepG2 cells in spiked human serum samples with excellent recoveries.Graphical abstract

Amplification-free and label-free rapid detection of Staphylococcus aureus using solution-gated graphene transistor-based DNA biosensor with hybridization enhancement by interface engineering

Staphylococcus aureus (S. aureus) is one of the leading causes of foodborne illnesses worldwide, posing a significant risk to food quality and safety. However, conventional detection methods often require complex amplification and labeling procedures, which are time consuming and expensive. In this study, an extremely sensitive electrochemical biosensor using a solution-gated graphene transistor (SGGT) was constructed for the amplification-free and label-free rapid detection of S. aureus by utilizing specific single-stranded DNA (ssDNA) to modify the gold (Au) sensing gate. This modified ssDNA was designed to hybridize with the target S. aureus DNA sequence. Interface engineering of the ssDNA-modified sensing gate via optimization of specific target and probe sequences was proposed to improve its hybridization efficiency. This enabled the sensitive quantitative detection of S. aureus DNA. The resulting biosensor exhibited excellent specificity in distinguishing non-complementary, 3-base/9-base DNA mismatch targets of S. aureus DNA and the specific DNA sequences of other common pathogenic bacteria. It also enabled the rapid detection of extracted target DNA sequences in solution (within 27 min) and had a low limit of detection (LoD) of 10−17 M over a wide linear detection range (10−17–10−8 M). Furthermore, the newly-developed biosensor facilitated the real-time and on-site detection of the S. aureus genome (ATCC 6538) with LoD of 10−17 M (103 CFU/mL) in a portable smart-sensing system. This suggests a promising future for the development of rapid, label-free, and amplification-free detection of foodborne illnesses using low-cost, highly sensitive SGGT DNA biosensors with appropriate functionalization and interface engineering of the sensing electrode.

A portable deep-learning-assisted digital single-particle counting biosensing platform for amplification-free nucleic acid detection using a lens-free holography microscope

The digital single-particle assay has emerged as a highly promising technology in various detection applications, such as food safety inspection, environmental monitoring, and in vitro diagnosis. Conventional digital assays rely on pre-amplification and expensive equipment, which limits their practical applications to point-of-care testing. Herein, we report a deep-learning-assisted digital single-particle counting biosensing platform for nucleic acid detection without pre-amplification using a portable and low-cost lens-free holography microscope. This device can perceive the number change of signal probes and capture microsphere probe holograms, which is ultra-lightweight (∼ 318 g), and has a low cost (∼ $70) and an ultrawide field of view of 24.396 mm2. The improved YOLOv7-based deep learning algorithm is trained to detect small objects (∼ 10 μm) in high-resolution images with high throughput. As a proof of concept, our strategy has successfully distinguished viable and nonviable Salmonella typhimurium quantitatively with high sensitivity (72 CFU/mL) without pre-amplification using phage-mediated DNA extraction and has been verified in various real samples. It has also been successfully applied to detecting non-nucleic acid targets in real samples, including procalcitonin and chloramphenicol. As a versatile and multi-functional platform, this platform exhibits excellent potential for point-of-care multi-type target detection in resource-limited settings.

CRISPR/Cas12a powered air-displacement enhanced evanescent wave fluorescence fiber-embedded microfluidic biochip for nucleic acid amplification-free detection of Escherichia coli O157:H7

Simple yet ultrasensitive and contamination-free quantification of environmental pathogenic bacteria is in high demand. In this study, we present a portable clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) powered Air-displacement enhanced Evanescent wave fluorescence Fiber-embedded microfluidic Biochip (AEFB) for the high-frequency and nucleic acid amplification-free ultrasensitive detection of Escherichia coli O157:H7. The performance of AEFB was dramatically enhanced upon employing a simple air-solution displacement process. Theoretical assays demonstrated that air-solution displacement significantly enhances evanescent wave field intensity on the fiber biosensor surface and increases the V-number in tapered fiber biosensors. Consequently, light-matter interaction is strengthened, and fluorescence coupling and collection efficiency are improved, considerably enhancing sensitivity. By integrating the CRISPR biosensing mechanism, AEFB facilitated rapid, accurate, nucleic acid amplification-free detection of E.coli O157:H7 with polymerase chain reaction (PCR)-level sensitivity (176 cfu/mL). To validate its practicality, AEFB was used to detect E.coli O157:H7 in surface water and wastewater. Comparison with RT-PCR showed a strong linear relationship (R2 = 0.9871), indicating the excellent accuracy and reliability of this technology in real applications. AEFB is highly versatile and can be easily extended to detect other pathogenic bacteria, which will significantly promote the high-frequency assessment and early-warning of bacterial contamination in aquatic environments.

Ultra-stable and highly-bright CsPbBr3 perovskite/silica nanocomposites for miRNA detection based on digital single-nanoparticle counting

Single-nanoparticle counting (SNPC) based on fluorescent tag (FT) stands out for its capacity to achieve amplification-free and sensitive detection of biomarkers. The stability and luminescence of FT are important to the sensitivity and reliability of SPNC. In this work, we developed novel perovskite/silica nanocomposites by in-situ nanoconfined growth of CsPbBr3 nanocrystals inside mesoporous structure of silica nanoparticles. PbBr(OH) was formed in an alkaline-assisted reaction triggered by water on the surface of CsPbBr3 nanocrystals. The as-obtained nanocomposites, featuring dual protection from silica matrix and PbBr(OH), exhibited high absolute photoluminescence quantum yield (PLQY) of 86.5% and demonstrated outstanding PL stability confronting with water, heat, ultrasound and UV-irradiation, which is desired by SNPC-based biosensor. Thereafter, these nanocomposites were used to construct an operationally friendly SNPC assay for the amplification-free quantification of cancer-associated miRNA. Quantitative detection of miRNA could be accomplished by directly counting the number of nanocomposites using a flow cytometer in this assay. This strategy did not ask for multiple washing steps and demonstrated specific and sensitive detection of miRNA 21, which exhibited a dynamic range of 1–1000 pM and limit of detection of 79 amol. The employment of highly stable perovskite/silica nanocomposites improved the test reliability and stability of SNPC, revealing the vast potential of perovskites in biosensing.