Unveiling Cas12j Trans-Cleavage Activity for CRISPR Diagnostics: Application to miRNA Detection in Lung Cancer Diagnosis.

Cas12j, a hypercompact and efficient Cas protein, has potential for use in CRISPR diagnostics, but has not yet been used because the trans-cleavage activity of Cas12j is veiled. Here, the trans-cleavage behavior of Cas12j1, 2, and 3 variants and evaluate their suitability for nucleic acid detection is unveiled. The target preferences and mismatch specificities of the Cas12j variants are precisely investigated and the optimal Cas12j reaction conditions are determined. As a result, the EXP-J assay for miRNA detection by harnessing the robust trans-cleavage activity of Cas12j on short ssDNA is developed. The EXP-J method demonstrates exceptional detection capabilities for miRNAs, proving that Cas12j can be a pivotal component in molecular diagnostics. Furthermore, the translational potential of the EXP-J assay is validated by detecting oncogenic miRNAs in plasma samples from lung cancer patients. This investigation not only elucidates the trans-cleavage characteristics of Cas12j variants, but also advances the Cas12j-based diagnostic toolkit.

Nanoplasmonic microarray-based solid-phase amplification for highly sensitive and multiplexed molecular diagnostics: application for detecting SARS-CoV-2.

A novel approach is introduced using nanoplasmonic microarray-based solid-phase recombinase polymerase amplification (RPA) that offers high sensitivity and multiplexing capabilities for gene detection. Nanoplasmonic microarrays were developed through one-step immobilization of streptavidin/biotin primers and fine-tuning the amplicon size to achieve high plasmon-enhanced fluorescence (PEF) on the nanoplasmonic substrate, thereby improving sensitivity. The specificity and sensitivity of solid-phase RPA on nanoplasmonic microarrays was evaluated in detecting E, N, and RdRP genes of SARS-CoV-2. High specificity was achieved by minimizing primer-dimer formation and employing a stringent washing process and high sensitivity obtained with a limit of detection of four copies per reaction within 30 min. In clinical testing with nasopharyngeal swab samples (n = 30), the nanoplasmonic microarrays demonstrated a 100% consistency with the PCR results for detecting SARS-CoV-2, including differentiation of Omicron mutations BA.1 and BA.2. This approach overcomes the sensitivity issue of solid-phase amplification, as well as offers rapidity, high multiplexing capabilities, and simplified equipment by using isothermal reaction, making it a valuable tool for on-site molecular diagnostics.

Recent advancements in implantable neural links based on organic synaptic transistors.

The progress of brain synaptic devices has witnessed an era of rapid and explosive growth. Because of their integrated storage, excellent plasticity and parallel computing, and system information processing abilities, various field effect transistors have been used to replicate the synapses of a human brain. Organic semiconductors are characterized by simplicity of processing, mechanical flexibility, low cost, biocompatibility, and flexibility, making them the most promising materials for implanted brain synaptic bioelectronics. Despite being used in numerous intelligent integrated circuits and implantable neural linkages with multiple terminals, organic synaptic transistors still face many obstacles that must be overcome to advance their development. A comprehensive review would be an excellent tool in this respect. Therefore, the latest advancements in implantable neural links based on organic synaptic transistors are outlined. First, the distinction between conventional and synaptic transistors are highlighted. Next, the existing implanted organic synaptic transistors and their applicability to the brain as a neural link are summarized. Finally, the potential research directions are discussed.

Dual structure-switching aptamer-mediated signal amplification cascade for SARS-CoV-2 detection.

Since the outbreak of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) at the end of 2019, the spread of the virus has posed a significant threat to public health and the global economy. This work proposed a one-step, dual-structure-switching aptamer-mediated signal amplification cascade for rapid and sensitive detection of the SARS-CoV-2 nucleocapsid protein. This system consisted of two DNA aptamers with structure-switching functionality and fuel DNA, where a cascade of strand hybridization and displacement triggered fluorescence generation and signal amplification. This aptamer-based amplification cascade required neither an amplification stage using enzymes nor pre-processing steps such as washing, viral isolation, and gene extraction. The assay could distinguish SARS-CoV-2 from other respiratory viruses and detect up to 1.0 PFU/assay of SARS-CoV-2 within 30 min at room temperature. In 35 nasopharyngeal clinical samples, the assay accurately assessed 25 positive and 10 negative clinical swab samples, which were confirmed using quantitative polymerase chain reaction. The strategy reported herein can help detect newly emerging pathogens and biomarkers of various diseases in liquid samples. In addition, the developed detection system consisting of only DNA and fluorophores can be widely integrated into liquid biopsy platforms for disease diagnosis.

CRISPR/Cas12a antifouling nanocomposite electrochemical biosensors enable amplification-free detection of Monkeypox virus in complex biological fluids.

The escalating global threat of infectious diseases, including monkeypox virus (MPXV), necessitates advancements in point-of-care diagnostics, moving beyond the constraints of conventional methods tethered to centralized laboratories. Here, we introduce multiple CRISPR RNA (crRNA)-based biosensors that can directly detect MPXV within 35 minutes without pre-amplification, leveraging the enhanced sensitivity and antifouling attributes of the BSA-based nanocomposite. Multiple crRNAs, strategically targeting diverse regions of the F3L gene of MPXV, are designed and combined to amplify Cas12a activation and its collateral cleavage of reporter probes. Notably, our electrochemical sensors exhibit the detection limit of 669 fM F3L gene without amplification, which is approximately a 15-fold improvement compared to fluorescence detection. This sensor also shows negligible changes in peak current after exposure to complex biological fluids, such as whole blood and serum, maintaining its sensitivity at 682 fM. This sensitivity is nearly identical to the conditions when only the F3L gene was present in PBS. In summary, our CRISPR-based electrochemical biosensors can be utilized as a high-performance diagnostic tool in resource-limited settings, representing a transformative leap forward in point-of-care testing. Beyond infectious diseases, the implications of this technology extend to various molecular diagnostics, establishing itself as a rapid, accurate, and versatile platform for detection of target analytes.

Multifunctional self-priming hairpin probe-based isothermal nucleic acid amplification and its applications for COVID-19 diagnosis.

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings.

Biporous silica nanostructure-induced nanovortex in microfluidics for nucleic acid enrichment, isolation, and PCR-free detection.

Efficient pathogen enrichment and nucleic acid isolation are critical for accurate and sensitive diagnosis of infectious diseases, especially those with low pathogen levels. Our study introduces a biporous silica nanofilms-embedded sample preparation chip for pathogen and nucleic acid enrichment/isolation. This chip features unique biporous nanostructures comprising large and small pore layers. Computational simulations confirm that these nanostructures enhance the surface area and promote the formation of nanovortex, resulting in improved capture efficiency. Notably, the chip demonstrates a 100-fold lower limit of detection compared to conventional methods used for nucleic acid detection. Clinical validations using patient samples corroborate the superior sensitivity of the chip when combined with the luminescence resonance energy transfer assay. The enhanced sample preparation efficiency of the chip, along with the facile and straightforward synthesis of the biporous nanostructures, offers a promising solution for polymer chain reaction-free detection of nucleic acids.

Micrometer-thick and porous nanocomposite coating for electrochemical sensors with exceptional antifouling and electroconducting properties.

Development of coating technologies for electrochemical sensors that consistently exhibit antifouling activities in diverse and complex biological environments over extended time is vital for effective medical devices and diagnostics. Here, we describe a micrometer-thick, porous nanocomposite coating with both antifouling and electroconducting properties that enhances the sensitivity of electrochemical sensors. Nozzle printing of oil-in-water emulsion is used to create a 1 micrometer thick coating composed of cross-linked albumin with interconnected pores and gold nanowires. The layer resists biofouling and maintains rapid electron transfer kinetics for over one month when exposed directly to complex biological fluids, including serum and nasopharyngeal secretions. Compared to a thinner (nanometer thick) antifouling coating made with drop casting or a spin coating of the same thickness, the thick porous nanocomposite sensor exhibits sensitivities that are enhanced by 3.75- to 17-fold when three different target biomolecules are tested. As a result, emulsion-coated, multiplexed electrochemical sensors can carry out simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, antigen, and host antibody in clinical specimens with high sensitivity and specificity. This thick porous emulsion coating technology holds promise in addressing hurdles currently restricting the application of electrochemical sensors for point-of-care diagnostics, implantable devices, and other healthcare monitoring systems.

A CRISPR/Cas12 trans-cleavage reporter enabling label-free colorimetric detection of SARS-CoV-2 and its variants.

We present a label-free colorimetric CRISPR/Cas-based method enabling affordable molecular diagnostics for SARS-CoV-2. This technique utilizes 3,3'-diethylthiadicarbocyanine iodide (DISC2(5)) which exhibits a distinct color transition from purple to blue when it forms dimers by inserting into the duplex of the thymidine adenine (TA) repeat sequence. Loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) was used to amplify target samples, which were subsequently subjected to the CRISPR/Cas12a system. The target amplicons would activate Cas12a to degrade nearby TA repeat sequences, preserving DISC2(5) in its free form to display purple as opposed to blue in the absence of the target. Based on this design approach, SARS-CoV-2 RNA was colorimetrically detected very sensitively down to 2 copies/µL, and delta and omicron variants of SARS-CoV-2 were also successfully identified. The practical diagnostic utility of this method was further validated by reliably identifying 179 clinical samples including 20 variant samples with 100% clinical sensitivity and specificity. This technique has the potential to become a promising CRISPR-based colorimetric platform for molecular diagnostics of a wide range of target pathogens.

ANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria.

Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.

3D printed fluidic swab for COVID-19 testing with improved diagnostic yield and user comfort.

The current standard method of diagnosing coronavirus disease 2019 (COVID-19) involves uncomfortable and invasive nasopharyngeal (NP) sampling using cotton swabs (CS), which can be unsuitable for self-testing. Although mid-turbinate sampling is an alternative, it has a lower diagnostic yield than NP sampling. Nasal wash (NW) has a similar diagnostic yield to NP sampling, but is cumbersome to perform. In this study, we introduce a 3D printed fluidic swab (3DPFS) that enables easy NW sampling for COVID-19 testing with improved diagnostic yield. The 3DPFS comprises a swab head, microchannel, and socket that can be connected to a syringe containing 250 µL of NW solution. The 3DPFS efficiently collects nasal fluid from the surface of the nasal cavity, resulting in higher sensitivity than CS for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This was confirmed by both reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and lateral flow assays (LFA) in virus-spiked nasal samples and clinical samples. Additionally, users reported greater comfort when using the 3DPFS compared to CS. These findings suggest that the 3DPFS can improve the performance of COVID-19 testing by facilitating efficient and less painful nasal sample collection.

Charge-shifting polyplex as a viral RNA extraction carrier for streamlined detection of infectious viruses.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, user-friendly nucleic acid testing that involves simple but efficient RNA extraction. Here, we present a charge-shifting polyplex as an RNA extraction carrier for advanced diagnosis of infectious viral diseases. The polyplex comprises poly(2-(dimethylamino) ethyl acrylate) (pDMAEA) electrostatically conjugated with RNA. The pDMAEA film can rapidly dissolve in the viral RNA solution, promoting immediate binding with RNA to form the polyplex, which enables the efficient capture of a substantial quantity of RNA. Subsequently, the captured RNA can be readily released by the quick hydrolysis of pDMAEA at the onset of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), streamlining the entire process from RNA extraction to analysis. The developed method requires only 5 min of centrifugation and enables the detection of RNA in a one-pot setup. Moreover, the proposed method is fully compatible with high-speed qRT-PCR kits and can identify clinical samples within 1 h including the entire extraction to detection procedure. Indeed, the method successfully detected influenza viruses, SARS-CoV-2, and their delta and omicron variants in 260 clinical samples with a sensitivity of 99.4% and specificity of 98.9%. This rapid, user-friendly polyplex-based approach represents a significant breakthrough in molecular diagnostics.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter.

Herein, we described a washing- and label-free clustered regularly interspaced short palindromic repeats (CRISPR)/LwaCas13a-based RNA detection method utilizing a personal glucose meter (PGM), which relies on the trans-cleavage activity of CRISPR/Cas13a and kinase reactions. In principle, the presence of target RNA activates the trans-cleavage of CRISPR/Cas13a, generating 2',3'-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.

Dual-mode SERS-based lateral flow assay strips for simultaneous diagnosis of SARS-CoV-2 and influenza a virus.

Since COVID-19 and flu have similar symptoms, they are difficult to distinguish without an accurate diagnosis. Therefore, it is critical to quickly and accurately determine which virus was infected and take appropriate treatments when a person has an infection. This study developed a dual-mode surface-enhanced Raman scattering (SERS)-based LFA strip that can diagnose SARS-CoV-2 and influenza A virus with high accuracy to reduce the false-negative problem of the commercial colorimetric LFA strip. Furthermore, using a single strip, it is feasible to detect SARS-CoV-2 and influenza A virus simultaneously. A clinical test was performed on 39 patient samples (28 SARS-CoV-2 positives, 6 influenza A virus positives, and 5 negatives), evaluating the clinical efficacy of the proposed dual-mode SERS-LFA strip. Our assay results for clinical samples show that the dual-mode LFA strip significantly reduced the false-negative rate for both SARS-CoV-2 and influenza A virus.

Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Ligation-free isothermal nucleic acid amplification.

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

A Novel Device for Blood Drainage after Le Fort I Osteotomy: Maxillary Sinus Ventilation Drainage (MSVD).

The purpose of this study is to present a novel maxillary sinus ventilation drainage (MSVD) device which facilitates blood drainage and nasal breathing after Le Fort I osteotomy. One hundred patients who underwent bimaxillary orthognathic surgery from January 2016 to June 2016 at the Department of Oral and Maxillofacial Surgery, Chung-Ang University Hospital were retrospectively selected and divided into two groups. MSVD was applied in 50 patients, who were allocated to the MSVD group, while the remaining 50 patients, in whom MSVD was not applied, were allocated to the non-MSVD group. All patients underwent a cone-beam computed tomography (CBCT) scan before and 2 days after surgery. CBCT was used to analyze middle meatus patency and the percentage of hematoma volume per entire maxillary sinus volume. Statistical comparisons between the two groups were performed using the Chi-squared and Mann-Whitney U tests to investigate the clinical effectiveness of MSVD. The MSVD group showed significantly higher maintenance ratio of the middle meatus patency and a higher percentage of maxillary sinus air volume (p < 0.05) than the non-MSVD group. MSVD facilitated nasal breathing after Le Fort I osteotomy by reducing hematoma inside the maxillary sinus and promoting middle meatal patency.

Surface-enhanced Raman scattering-based immunoassay for severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected humans worldwide for over a year now. Although various tests have been developed for the detection of SARS-CoV-2, advanced sensing methods are required for the diagnosis, screening, and surveillance of coronavirus disease 2019 (COVID-19). Here, we report a surface-enhanced Raman scattering (SERS)-based immunoassay involving an antibody pair, SERS-active hollow Au nanoparticles (NPs), and magnetic beads for the detection of SARS-CoV-2. The selected antibody pair against the SARS-CoV-2 antigen, along with the magnetic beads, facilitates the accurate direct detection of the virus. The hollow Au NPs exhibit strong, reproducible SERS signals, allowing sensitive quantitative detection of SARS-CoV-2. This assay had detection limits of 2.56 fg/mL for the SARS-CoV-2 antigen and 3.4 plaque-forming units/mL for the SARS-CoV-2 lysates. Furthermore, it facilitated the identification of SARS-CoV-2 in human nasopharyngeal aspirates and diagnosis of COVID-19 within 30 min using a portable Raman device. Thus, this assay can be potentially used for the diagnosis and prevention of COVID-19.