Sensitive DNA Detection Research Articles

Which 2D Material is Better for DNA Detection: Graphene, MoS2, or MXene?

Despite much research on characterizing 2D materials for DNA detection with nanopore technology, a thorough comparison between the performance of different 2D materials is currently lacking. In this work, using extensive molecular dynamics simulations, we compare nanoporous graphene, MoS2 and titanium carbide MXene (Ti3C2) for their DNA detection performance and sensitivity. The ionic current and residence time of DNA are characterized in each nanoporous materials by performing hundreds of simulations. We devised two statistical measures including the Kolmogorov-Smirnov test and the absolute pairwise difference to compare the performance of nanopores. We found that graphene nanopore is the most sensitive membrane for distinguishing DNA bases. The MoS2 is capable of distinguishing the A and T bases from the C and G bases better than graphene and MXene. Physisorption and the orientation of DNA in nanopores are further investigated to provide molecular insight into the performance characteristics of different nanopores.

Carbon quantum dots functionalized tapered optical fiber for highly sensitive and specific detection of Leptospira DNA

In this paper, we report on the development of DNA biosensor using carbon quantum dots (CQDs) to enhance Leptospira DNA detection. Leptospira is a bacterium that invades human body and cause leptospirosis disease. Leptospirosis has attracted attention mainly due to several cases of human mortality which have distressed health professionals. This biosensor system employs tapered optical fiber as a sensing transducer. The CQDs were functionalized at the tapered region via in situ deposition (drop-casted) before incubation using commercialized chemicals to link the 16 s ribosomal RNA gene (rrs) probe DNA. Detection was achieved with the occurrence of hybridization between the probe DNA and different concentrations of complementary DNA (Leptospira DNA). This biosensor with limit of detection (LOD) of 1.0 fM demonstrated better sensitivity at 1.8295 nm/nM and higher affinity as its dissociation constant (Kd) of 0.552 × 10−09 M is lower compared to previous reported biosensors without CQDs. High specificity biosensor is validated when minimal shift was observed for non-complementary DNA. This novel biosensor was also able to distinguish between genomic DNA of Leptospira serovars and Clostridium difficile as control sample. The proposed biosensor has successfully exhibited the prominent role of nanomaterial for sensor enhancement. Consequently, the biosensor approach in this study has emphasized the potential for the development of more sensitive and reliable leptospirosis diagnostic testing in the future.

Varicella zoster-associated progressive lower cranial and upper cervical polyneuropathy: a case report

BackgroundMultiple cranial neuropathies carry a wide range of differential diagnoses, and when combined with cerebrospinal fluid monocytosis they often suggest an infective etiology. Reactivation of varicella zoster virus has been associated with a wide range of neurological complications. Among the cranial nerves, the upper cranial nerves (trigeminal and facial nerves) are more commonly affected; there have been some reports of lower cranial polyneuropathies resulting from varicella zoster virus reactivation. However, polyneuropathy involving both the cranial and cervical nerves is rarely reported.Case presentationThis report highlights the case of a 64-year-old Chinese man presenting with an acute, severe dysphagia and dysphonia secondary to herpes zoster-associated progressive polyneuropathy involving the lower cranial and upper cervical nerves, without any mucocutaneous manifestations.ConclusionsTo our knowledge, this is the first case of varicella zoster virus-associated cranial and cervical polyneuropathy in the literature. The report also highlights the poor sensitivity of varicella zoster virus DNA detection by polymerase chain reaction in the cerebrospinal fluid, and proposes that serology be routinely performed in such polymerase chain reaction-negative cases without a clear diagnosis.

Dual phenomenon of surface plasmon and laser optics induced EM enhancement for rapid DNA detection with semiconductor nanostructures (SiNWs/AgNPs)

Recently exploring bioanalytical instrumentation principles emphasized with combination of optics and EM fields in surface enhanced Raman spectroscopy (SERS). The principles favour the rapid detection of DNA with proper fabrication of SERS sensor. 1D semiconductor nanostructures (SiNWs) functionalization with plasmonic silver nanoparticles are fabricated by PLD process and exploited for SERS detection of DNA. The DNA nucleotide bands are indexed with reference to their Raman shift as Adenine (A), Guanine (G), Cytosine (C), Thymine (T) at Raman spectral regions of 1352 cm-1, 676 cm-1, 1625 cm-1, 1441 cm-1 respectively. The finest conditions for sensitive detection of DNA were investigated and their interaction mechanism are presented. The point-to-point reproducibility measurements imprinted higher degree of concordancy. The designed SERS sensor will be definitely helpful almost in all application fields with portable handheld Raman spectrophotometer in near future.

Rapid and Highly Sensitive Detection of Target DNA Related to COVID-19 Virus With a Fluorescent Bio-conjugated Probe via a FRET Mechanism

A novel cyanine 3 (Cy3)-based bio-conjugated sensor has been developed to detect target DNA or extracted RNA from COVID -19 samples using the fluorescence resonance energy transfer (FRET) experiment. A special sequence of the COVID -19 genome was selected as a complementary DNA (target DNA) part. The opposite chain of this target sequence was designed in 2 parts; one part was attached to the Cy3 organic dye (capture DNA or Cy3- DNA), and the other part was attached to the BHQ2 molecule (quencher DNA or BHQ2- DNA). The Cy3 molecule acts as a donor pair, and BHQ2 acts as an acceptor pair in the FRET experiment. The capture DNA and quencher DNA can form a sandwiched complex in the presence of target DNA. The formation of the entitled sandwiched hybrid causes the decrement of emission intensity of the Cy3 donor in bio-conjugated Cy3-DNA via energy transfer from Cy3 (as a donor) to BHQ2 (as an acceptor). Indeed, in the presence of non-complementary DNA, the pairing of DNA strands does not occur, the FRET phenomenon does not exist, and therefore fluorescence intensity of Cy3 does not decrease. Moreover, this biosensor was successfully applied to analyze real samples containing extracted RNA of COVID -19 prepared for the reverse transcriptase-polymerase chain reaction (RT-PCR) test, and the results were promising.

A highly-efficient 3D DNAzyme motor for sensitive biosensing analysis

Herein, driven by the need of highly-efficient DNAzyme-amplified detection strategy, a novel 3D DNAzyme motor was designed as a biosensor platform for realizing sensitive detection of target DNA. The 3D DNAzyme motor was composed of target-activated DNAzyme nanowires and substrates H1-Fc that co-immobilized on Au@Fe3O4 nanoparticles (Au@Fe3O4NPS) surface, possessing high local concentration of DNA reactants and shortened distance between DNAzyme and substrates for enhancing electrochemical signal. Compared with traditional DNAzyme-powered machines, the target-activated DNAzyme nanowires of 3D DNAzyme motor had greater flexibility and more powerful cleavage capability without troublesome sequence optimization, which overcame the space limitation and simultaneously interacted with adjacent and distant substrates H1-Fc to output a large amount of cleavage products with high signal response. Therefore, on account of the above-mentioned merits of nanoparticles localization DNA design and DNAzyme nanowires, the reported 3D DNAzyme motor ingeniously overcame many defects existing in traditional DNAzyme-amplified detection strategies such as low reactants concentration, limited flexibility of DNAzyme and small DNAzyme swing range, realizing the sensitive detection of target DNA with a detection limit of 1.7 fM ranging from 5 fM to 50 nM. Impressively, the 3D DNAzyme motor here presented a new strategy to achieve effective DNAzyme signal amplification and provided a reference for the assembly of various and functional 3D DNA machines in the future.

Early Detection of the Spinach Downy Mildew Pathogen in Leaves by Recombinase Polymerase Amplification.

Downy mildew of spinach, caused by Peronospora effusa, is a major economic threat to both organic and conventional spinach production. Symptomatic spinach leaves are unmarketable and spinach with latent infections are problematic because symptoms can develop postharvest. Therefore, early detection methods for P. effusa could help producers identify infection before visible symptoms appear. Recombinase polymerase amplification (RPA) provides sensitive and specific detection of pathogen DNA and is a rapid, field-applicable method that does not require advanced technical knowledge or equipment-heavy DNA extraction. Here, we used comparative genomics to identify a unique region of the P. effusa mitochondrial genome to develop an RPA assay for the early detection of P. effusa in spinach leaves. In tandem, we established a TaqMan quantitative PCR (qPCR) assay and used this assay to validate the P. effusa specificity of the locus across Peronospora spp. and to compare assay performance. Neither the TaqMan qPCR nor the RPA showed cross reactivity with the closely related beet downy mildew pathogen, P. schachtii. TaqMan qPCR and RPA have detection thresholds of 100 and 900 fg of DNA, respectively. Both assays could detect P. effusa in presymptomatic leaves, with RPA-based detection occurring as early as 5 days before the appearance of symptoms and TaqMan qPCR-based detection occurring after 24 h of plant exposure to airborne spores. Implementation of the RPA detection method could provide real-time information for point-of-care management strategies at field sites.

A stable Au–N bond controlled probe immobilization approach for the sensitive detection of kirsten rat sarcoma viral oncogene DNA using NH2-HMS@Au

A stable Au–N bond controlled probe immobilization approach for the sensitive detection of kirsten rat sarcoma viral oncogene DNA using NH2-HMS@Au

A Highly Sensitive and Specific Detection Method for Mycobacterium tuberculosis Fluoroquinolone Resistance Mutations Utilizing the CRISPR-Cas13a System.

ObjectivesCRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear.MethodsA combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed.ResultsA set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated Mycobacterium tuberculosis strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated M. tuberculosis, the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays.ConclusionThese results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.

An ultrafast ratiometric electrochemical biosensor based on potential-assisted hybridization for nucleic acids detection

The development of rapid nucleic acid detection methods, which are promising the diagnostic standard of the infectious disease, could expand more options for disease traceability and controllability. Nucleic acid hybridization-based biosensing techniques still encounter limitations in meeting the requirements for rapid detection. Therefore, we proposed a potential-assisted ratiometric electrochemical biosensor for rapid and accurate target DNA detection. This dual-signal analysis system actuated an enzyme-free detection process. The application of an external electric field accelerated molecular dynamics, resulting in highly efficient collision chances. This hybridization process was thus improved from hours to minutes compared with passive hybridization approach. The biosensor not only had a high sensitivity with the detection limit of 12 fM, but also features a robust capability in identifying single-base mismatch. Moreover, the biosensor realized sensitive detection of target DNA in complex biological environments. Overall, this sensing strategy exhibits a promising potential for application in point-of-care testing.

Alinity m, a Random-Access System, for Hepatitis B Virus DNA Quantification in Plasma and Whole Blood Collected on Dried Blood Spots.

ABSTRACTThe International Liver Association recommends the use of accurate and sensitive molecular methods for determination of hepatitis B virus (HBV) DNA levels in plasma or serum of chronic HBsAg carriers. The level of HBV replication represents the strongest predictive biomarker associated with disease progression and long-term outcome of chronic HBV infection. The purpose of this study was to evaluate the ability to the new Alinity m System to detect and quantify HBV DNA in plasma and whole blood collected on dried blood spots (DBS). Paired plasma and DBS samples from patients chronically infected with various HBV genotypes were tested in parallel for HBV DNA detection and quantification. There is a linear relationship between HBV DNA levels measured in plasma samples using the Alinity m HBV assay and the Xpert HBV viral load assay, used for comparison. A slight deviation (0.03 ± 0.31 log IU/mL) was observed within the quantitative range. In DBS, HBV DNA levels closely correlated with levels measured in plasma. All patients had detectable and quantifiable HBV DNA by DBS testing, except for one patient with a plasma HBV DNA level above 2,000 IU/mL. In conclusion, the newly developed real-time PCR-based assay Alinity m HBV assay can correctly detect HBV DNA in DBS, especially for patients with blood HBV DNA levels above 2,000 IU/mL, and also accurately quantify HBV DNA in plasma samples.IMPORTANCE Hepatitis B virus is one of the most prevalent blood-borne viruses affecting the liver and causing acute and chronic hepatitis. Only a small proportion of people with HBV infection are diagnosed. HBV DNA measurement is critical in clinical practice for the diagnosis and treatment decisions of patients requiring antiviral therapy. Dried blood spot (DBS) collection provides a simple, practical, and acceptable alternative to venous blood collection, especially in community settings. We have demonstrated high sensitivity and specificity for HBV DNA detection in DBS compared to plasma samples, especially when using clinically relevant cutoffs of 2,000 and 20,000 IU/mL. Results support the use of DBS in community-based settings.

Organometallic hotspot engineering for ultrasensitive EC-SERS detection of pathogenic bacteria-derived DNAs

The sensitivity and limit-of-detection (LOD) of the traditional surface-enhanced Raman spectroscopy (SERS) platform suffer from the requirement of precise positioning of small analytes, including DNAs and bacteria, into narrow hotspots. In this study, a novel SERS sensor was developed using electrochemical deposition onto metal nanopillars (ECOMPs) combined with complementary DNAs (cDNAs) for the detection of pathogenic bacteria. Applying a redox potential to AuCl4− ions actively engineered the organometallic hotspots based on the cDNAs in a short time (<10 min) and simultaneously produced SERS signals. Because of the influence of potential-driven morphological properties on the SERS efficiency in the cDNA domains and the resonant coupling of internal fields with the fields confined between adjacent ECOMPs-cDNAs, the optimum growth time was determined to be 5 min. The EC-SERS detection and discrimination of Enterococcus faecium and Staphylococcus aureus were successfully carried out because of the DNA complementarity. Compared with plasmonic metal nanopillars (MPs)-cDNAs, the enhancement factor of the ECOMPs-cDNAs was estimated to be ∼2.0 × 103. A quantitative investigation revealed that a highly linear progression in the target DNA concentration range (0.05–100 nM) and a LOD of ∼0.035 nM were achieved. The specificity of the ECOMPs-cDNAs was validated by cross-hybridization. The platform was also used to assay human whole blood containing 0.1 nM bacterial DNAs. The proposed strategy provides the potential for highly sensitive SERS-based multiplex DNA detection in clinical diagnostics.

Machine-Learning-Based Clinical Biomarker Using Cell-Free DNA for Hepatocellular Carcinoma (HCC).

Simple SummaryCirculating cell-free DNA (cfDNA) has attracted a great deal of scientific interest as a predictive biomarker for the diagnosis and prognosis of hepatocellular carcinoma (HCC). HCC result in high mortality due to the absence of blood biomarkers for early diagnosis and prognosis. We established cfDHCC as a new scoring system by applying a machine learning algorithm that integrates the expression profiles of cfDNA. Based on this, it was possible to accurately predict the clinico-pathological characteristics of patients with HCC as well as improve their survival. (1) Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Although various serum enzymes have been utilized for the diagnosis and prognosis of HCC, the currently available biomarkers lack the sensitivity needed to detect HCC at early stages and accurately predict treatment responses. (2) Methods: We utilized our highly sensitive cell-free DNA (cfDNA) detection system, in combination with a machine learning algorithm, to provide a platform for improved diagnosis and prognosis of HCC. (3) Results: cfDNA, specifically alpha-fetoprotein (AFP) expression in captured cfDNA, demonstrated the highest accuracy for diagnosing malignancies among the serum/plasma biomarkers used in this study, including AFP, aspartate aminotransferase, alanine aminotransferase, albumin, alkaline phosphatase, and bilirubin. The diagnostic/prognostic capability of cfDNA was further improved by establishing a cfDNA score (cfDHCC), which integrated the total plasma cfDNA levels and cfAFP-DNA expression into a single score using machine learning algorithms. (4) Conclusion: The cfDHCC score demonstrated significantly improved accuracy in determining the pathological features of HCC and predicting patients’ survival outcomes compared to the other biomarkers. The results presented herein reveal that our cfDNA capture/analysis platform is a promising approach to effectively utilize cfDNA as a biomarker for the diagnosis and prognosis of HCC.

Plasmon-modulated fluorescence nanoprobes for enzyme-free DNA detection via target signal enhancement and off-target quenching

Rapid, sensitive, and reliable nucleic acid assay is crucial for the molecular diagnosis of many diseases. For high sensitivity, conventional techniques require time-consuming, high-cost, and complicated procedures, such as enzymatic gene amplification, labeling, and purification, limiting their applications to point-of-care diagnostics. Herein we report a new DNA nanoprobe based on the dual effects of target-specific plasmon-enhanced fluorescence and off-target plasmonic quenching. Janus gold half-shell/polystyrene nanospheres (hsAu/PSs, ∼150 nm diameter) are tethered with capture single-stranded DNA (ssDNA), coupled with a fluorophore-conjugated reporter ssDNA through sandwich-type hybridization with target DNA, resulting in 5-fold increase through plasmon-enhanced fluorescence. Smaller gold nanoparticles (∼13 nm diameter) are subsequently introduced as quenchers to reduce background fluorescence from unhybridized reporter ssDNA, increasing the sensitivity about 103 times. The limit of detection of the dual-mode plasmonic DNA nanoprobe is 16 pM at room temperature in 1 h for the target gene of Klebsiella pneumoniae carbapenemase. The nanoprobe also exhibits a high selectivity enough to discriminate a single-base difference in the target gene. Our strategy harnesses both of the plasmon-mediated fluorescence enhancement and quenching effects through the sophisticated design of nanoscale colloids, which opens a promising avenue to the enzyme-free, simple, sensitive, and selective detection of pathogenic DNA.

Sensitive photoelectrochemical detection of colitoxin DNA based on NCDs@CuO/ZnO heterostructured nanocomposites with efficient separation capacity of photo-induced carriers.

Ametal-organic framework (MOF) of Cu-TPA (terephthalic acid) microsphere was prepared, followed by calcinating the MOF precursor of Cu-TPA/ZIF-8 mixture to obtain the CuO/ZnO. N-doped carbon dots (NCDs) were employed to combine the CuO/ZnO composite to form a tripartite heterostructured architecture of NCDs@CuO/ZnO, which led to a fierce enlargement of the photocurrent response. This was ascribed to the thinner-shell structure of the CuO microsphere and the fact thathollow ZnO particles could sharply promote the incidence intensity of visible light. The more porous defectiveness exposed on CuO/ZnO surface was in favor of rapidly infiltrating electrolyte ions. The p-n type CuO/ZnO composite with more contact interface could abridge the transfer distance of photo-induced electron (e-1)/hole (h+) pairs and repress their recombination availably. NCDs not only could boost electron transfer rate on the electrode interface but also successfully sensitized the CuO/ZnO composite, which resulted in high conversion efficiency of photon-to-electron. The probe DNA (S1) was firmly assembled on the modified ITO electrode surface (S1/NCDs@CuO/ZnO) through an amidation reaction. Under optimal conditions, the prepared DNA biosensor displayed a wide linear range of 1.0 × 10-6 ~ 7.5 × 10-1nM and a low limit of detection (LOD) of 1.81 × 10-7nM for colitoxin DNA (S2) measure, which exhibited a better photoelectrochemistry (PEC) analysis performance than that obtained by differential pulse voltammetry techniques. The relative standard deviation (RSD) of the sensing platform for target DNA detection of 5.0 × 10-2nM was 6.3%. This proposed DNA biosensor also showed good selectivity, stability, and reproducibility, demonstrating that the well-designed and synthesized photoactive materials of NCDs@CuO/ZnO are promising candidates forPEC analysis.

Highly sensitive environmental DNA detection of topmouth gudgeon, Pseudorasbora parva: a comparison of qPCR and microfluidic qdPCR

Topmouth gudgeon is a freshwater fish species native to East Asia. Nowadays, P. parva is spread throughout Europe which is of concern because besides being considered one of the worst aquatic Invasive Alien Species (IAS) in Europe it is also a known vector of Spherotecum destruens, the rosette-like parasite lethal to other fish species. The present study describes the development and validation of a new species-specific assay based on hydrolysis probe chemistry to detect P. parva environmental DNA (eDNA) in water samples collected in a northern region of Italy (Friuli Venezia Giulia). Water samples were collected from 55 sites in an area where partial information on the occurrence of the species is available. eDNA was isolated from all samples and the presence of P. parva eDNA was tested by means of qPCR (quantitative PCR) and microfluidic qdPCR (quantitative digital PCR) techniques. Field results for both qPCR and qdPCR were largely in agreement in terms of detection (presence/absence). Thus, we judged the presence/absence by combining the results from the two methods and found that nine sites showed “strong positive” signal of P. parva eDNA (at least 2 positive replicates), 3 showed “suspected” (only 1 positive replicate), and 42 showed “absent”. The current study shows the strong potential of the newly developed eDNA approach to be a valuable addition to the monitoring of the highly invasive topmouth gudgeon in freshwater ecosystems.

Ultra-Highly Sensitive DNA Detection with Conducting Polymer-Modified Electrodes: Mechanism, Manufacture and Prospects for Rapid e-PCR

At low copy number, sequence detection by polymerase chain reaction (PCR) requires up to 30 cycles (amplification 109) to produce a reliably detectable concentration of fluorescently-labelled amplicons. The cycle number and hence detection time is determined by the analytical sensitivity of the detector. Hybridisation of complementary DNA strands to oligonucleotide-modified conducting polymer electrodes yields an increase in the charge transfer resistance for the ferri-ferrocyanide redox couple. We demonstrate sensors using screen-printed carbon electrodes modified with a conducting polymer formed from a monomer pre-functionalised with complementary oligonucleotide, with pM sensitivity for short sequences and aM for bacterial lysate, with a response time-scale of 5 min. The response is due to the variation of electrical resistance within the polymer film. We develop a mechanism based on repulsion from the solution interface of dopant anions by the charge associated with surface-bound DNA. With results for >160 single-use sensors, we formulate a response model based on percolation within a random resistor network and highlight challenges for large-scale manufacture of such sensors. Such sensors used for label-free electrochemical detection for PCR (e-PCR) would decrease the required cycle number from 30 to less than 10 and would offer a much simplified instrument construction.

Ultrasensitive Label-Free DNA Detection Based on Solution-Gated Graphene Transistors Functionalized with Carbon Quantum Dots.

Developing highly sensitive, reliable, cost-effective label-free DNA biosensors is challenging with traditional fluorescence, electrochemical, and other techniques. Most conventional methods require labeling fluorescence, enzymes, or other complex modification. Herein, we fabricate carbon quantum dot (CQD)-functionalized solution-gated graphene transistors for highly sensitive label-free DNA detection. The CQDs are immobilized on the surface of the gate electrode through mercaptoacetic acid with the thiol group. A single-stranded DNA (ssDNA) probe is immobilized on CQDs by strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, which led to a shift of Dirac voltage and the channel current response. The limit of detection can reach 1 aM which is 2-5 orders of magnitude lower than those of other methods reported previously. The sensor also exhibits a good linear range from 1 aM to 0.1 nM and has good specificity. It can effectively distinguish one-base mismatched target DNA. The response time is about 326 s for the 1 aM target DNA molecules. This work provides good perspectives on the applications in biosensors.

CLIPON: A CRISPR‐Enabled Strategy that Turns Commercial Pregnancy Test Strips into General Point‐of‐Need Test Devices

AbstractDesirable biosensing assays need to be sensitive, specific, cost‐effective, instrument‐free, and versatile. Herein we report a new strategy termed CLIPON (CRISPR and Large DNA assembly Induced Pregnancy strips for signal‐ON detection) that can deliver these traits. CLIPON integrates a commercial pregnancy test strip (PTS) with four biological elements: the human chorionic gonadotropin (hCG), CRISPR‐Cas12a, crRNA and cauliflower‐like large‐sized DNA assemblies (CLD). CLIPON uses the Cas12a/crRNA complex both to recognize a target of interest and to release CLD‐bound hCG so that target presence can translate into a colorimetric signal on the PTS. We demonstrate the versatility of CLIPON through sensitive and specific detection of HPV genomic DNA, SARS‐CoV‐2 genomic RNA and adenosine. We also engineer a cell phone app and a hand‐held microchip to achieve signal quantification. CLIPON represents an attractive option for biosensing and point‐of‐care diagnostics.

CLIPON: A CRISPR-Enabled Strategy that Turns Commercial Pregnancy Test Strips into General Point-of-Need Test Devices.

Desirable biosensing assays need to be sensitive, specific, cost-effective, instrument-free, and versatile. Herein we report a new strategy termed CLIPON (CRISPR and Large DNA assembly Induced Pregnancy strips for signal-ON detection) that can deliver these traits. CLIPON integrates a commercial pregnancy test strip (PTS) with four biological elements: the human chorionic gonadotropin (hCG), CRISPR-Cas12a, crRNA and cauliflower-like large-sized DNA assemblies (CLD). CLIPON uses the Cas12a/crRNA complex both to recognize a target of interest and to release CLD-bound hCG so that target presence can translate into a colorimetric signal on the PTS. We demonstrate the versatility of CLIPON through sensitive and specific detection of HPV genomic DNA, SARS-CoV-2 genomic RNA and adenosine. We also engineer a cell phone app and a hand-held microchip to achieve signal quantification. CLIPON represents an attractive option for biosensing and point-of-care diagnostics.