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1.
Small ; 20(13): e2307262, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37963850

RESUMO

Breast cancer (BC) is a major global health problem, with ≈20-25% of patients overexpressing human epidermal growth factor receptor 2 (HER2), an aggressive marker, yet access to early detection and treatment varies across countries. A low-cost, equipment-free, and easy-to-use polydiacetylene (PDA)-based colorimetric sensor is developed for HER2-overexpressing cancer detection, designed for use in low- and middle-income countries (LMICs). PDA nanoparticles are first prepared through thin-film hydration. Subsequently, hydrophilic magnetic nanoparticles and HER2 antibodies are sequentially conjugated to them. The synthesized HER2-MPDA can be concentrated and separated by a magnetic field while inheriting the optical characteristics of PDA. The specific binding of HER2 antibody in HER2-MPDA to HER2 receptor in HER2-overexpressing exosomes causes a blue-to-red color change by altering the molecular structure of the PDA backbone. This colorimetric sensor can simultaneously separate and detect HER2-overexpressing exosomes. HER2-MPDA can detect HER2-overexpressing exosomes in the culture medium of HER2-overexpressing BC cells and in mouse urine samples from a HER2-overexpressing BC mouse model. It can selectively isolate and detect only HER2-overexpressing exosomes through magnetic separation, and its detection limit is found to be 8.5 × 108 particles mL-1. This colorimetric sensor can be used for point-of-care diagnosis of HER2-overexpressing BC in LMICs.


Assuntos
Neoplasias da Mama , Compostos de Diazônio , Exossomos , Nanopartículas , Polímero Poliacetilênico , Piridinas , Humanos , Animais , Camundongos , Feminino , Colorimetria , Exossomos/metabolismo , Neoplasias da Mama/metabolismo , Anticorpos , Fenômenos Magnéticos
2.
Small ; 20(35): e2308317, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38564785

RESUMO

Proactive management of foodborne illness requires routine surveillance of foodborne pathogens, which requires developing simple, rapid, and sensitive detection methods. Here, a strategy is presented that enables the detection of multiple foodborne bacteria using a 3D nanostructure swab and deep learning-based Raman signal classification. The nanostructure swab efficiently captures foodborne pathogens, and the portable Raman instrument directly collects the Raman signals of captured bacteria. a deep learning algorithm has been demonstrated, 1D convolutional neural network with binary labeling, achieves superior performance in classifying individual bacterial species. This methodology has been extended to mixed bacterial populations, maintaining accuracy close to 100%. In addition, the gradient-weighted class activation mapping method is used to provide an investigation of the Raman bands for foodborne pathogens. For practical application, blind tests are conducted on contaminated kitchen utensils and foods. The proposed technique is validated by the successful detection of bacterial species from the contaminated surfaces. The use of a 3D nanostructure swab, portable Raman device, and deep learning-based classification provides a powerful tool for rapid identification (≈5 min) of foodborne bacterial species. The detection strategy shows significant potential for reliable food safety monitoring, making a meaningful contribution to public health and the food industry.


Assuntos
Aprendizado Profundo , Microbiologia de Alimentos , Nanoestruturas , Análise Espectral Raman , Análise Espectral Raman/métodos , Nanoestruturas/química , Doenças Transmitidas por Alimentos/microbiologia , Bactérias/isolamento & purificação
3.
Analyst ; 148(2): 316-327, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36484412

RESUMO

Most nucleic acid biosensors employ nucleic acid-processing enzymes to bind, degrade, splice, synthesize, and modify nucleic acids. Utilizing their unique substrate preference, binding mode, and catalytic activity is of great importance in designing nucleic acid biosensors. Combination with DNA-processing enzymes enables them to transform into a new generation of molecular diagnostics tools with enhanced selectivity and sensitivity and reduced reaction time. Here, we report an isothermal amplification strategy by coemploying a structure-specific endonuclease (flap endonuclease 1, FEN1) and a strand-displacing DNA polymerase (Bst DNA polymerase) to detect long RNA targets. This approach couples the FEN1-driven invasive cleavage reaction with toehold-mediated rolling circle amplification (iFEN-tRCA), enabling the highly selective and rapid detection of long RNA targets and offering a detection limit below 10 pM within 1 h. We used two targets, such as human epidermal growth factor receptor 2 (HER2, encoded by ERBB2) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP, encoded by PPP1R1B), associated with prognosis or response to anticancer therapy. We demonstrated the feasibility and quantitative capability of the iFEN-tRCA assay by assessing the expression of two RNA transcripts (ERBB2 and PPP1R1B) with total RNA extracts purified from human breast cancer cells. Therefore, we envision that the developed assay will provide a suitable prognostic and diagnostic tool for identifying appropriate patients for HER2-targeted therapy and predicting the clinical outcome and occurrence of metastasis relapse in breast cancer.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Transcriptoma , Técnicas de Amplificação de Ácido Nucleico , DNA/genética , DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , RNA
4.
Anal Chem ; 94(8): 3535-3542, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35061345

RESUMO

With the growth of drug-facilitated crimes, prevention has become increasingly important. Although various drug detection technologies exist, most focus on postconsumption detection. However, the prevention of drug-facilitated crimes requires technology for the quick and easy detection of amphetamine-type stimulants (ATSs) before ingestion. Herein, drug screening kits (DSKs) were developed for the simple detection of ATSs in drinks. The DSKs consisted of polydiacetylene nanofiber-based paper sensors fabricated by electrospinning with 10,12-pentacosadiynoic acid (PCDA) and PCDA-dopamine as sensing materials that can bind ATSs via hydrogen bonding and π-π interactions. Dropping a drink on the DSK provided an immediate visual indication of the presence of ATSs. When ATSs were present in the drink, the color of the DSK clearly changed from blue to red, with the increase in red intensity being more than twofold greater than that observed when water alone was tested. Notably, the result could be confirmed by the naked eye without any analytical instrumentation. A color change indicating the presence of ATSs was successfully observed in various alcoholic and nonalcoholic drinks. These results indicate the potential of DSKs for preventing drug-facilitated crimes caused by unwanted drug intake.


Assuntos
Estimulantes do Sistema Nervoso Central , Nanofibras , Anfetamina , Colorimetria/métodos
5.
Chem Eng J ; 442: 136143, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35382003

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a pandemic of acute respiratory disease, namely coronavirus disease (COVID-19). This disease threatens human health and public safety. Early diagnosis, isolation, and prevention are important to suppress the outbreak of COVID 19 given the lack of specific antiviral drugs to treat this disease and the emergence of various variants of the virus that cause breakthrough infections even after vaccine administration. Simple and prompt testing is paramount to preventing further spread of the virus. However, current testing methods, namely RT-PCR, is time-consuming. Binding of the SARS-CoV-2 spike (S) glycoprotein to human angiotensin-converting enzyme 2 (hACE2) receptor plays a pivotal role in host cell entry. In the present study, we developed a hACE2 mimic peptide beacon (COVID19-PEB) for simple detection of SARS-CoV-2 using a fluorescence resonance energy transfer system. COVID19-PEB exhibits minimal fluorescence in its ''closed'' hairpin structure; however, in the presence of SARS-CoV-2, the specific recognition of the S protein receptor-binding domain by COVID19-PEB causes the beacon to assume an ''open'' structure that emits strong fluorescence. COVID19-PEB can detect SARS-CoV-2 within 3 h or even 50 min and exhibits strong fluorescence even at low viral concentrations, with a detection limit of 4 × 103 plaque-forming unit/test. Furthermore, in SARS-CoV-2-infected patient samples confirmed using polymerase chain reaction, COVID19-PEB accurately detected the virus. COVID19-PEB could be developed as a rapid and accurate diagnostic tool for COVID-19.

6.
Sensors (Basel) ; 18(2)2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29462867

RESUMO

Aflatoxins (AFs) are highly toxic compounds that can cause both acute and chronic toxicity in humans. Aflatoxin B1 (AFB1) is considered the most toxic of AFs. Therefore, the rapid and on-site detection of AFB1 is critical for food safety management. Here, we report the on-site detection of AFB1 in grains by a portable surface plasmon resonance (SPR) sensor. For the detection of AFB1, the surface of an SPR Au chip was sequentially modified by cysteine-protein G, AFB1 antibody, and bovine serum albumin (BSA). Then, the sample solution and AFB1-BSA conjugate were flowed onto the Au chip in serial order. In the absence of AFB1, the SPR response greatly increased due to the binding of AFB1-BSA on the Au chip. In the presence of AFB1, the SPR response showed little change because the small AFB1 molecule binds on the Au chip instead of the large AFB1-BSA molecule. By using this portable SPR-based competitive immunoassay, the sensor showed low limits of detection (2.51 ppb) and quantification (16.32 ppb). Furthermore, we successfully detected AFB1 in rice, peanut, and almond samples, which suggests that the proposed sensing method can potentially be applied to the on-site monitoring of mycotoxins in food.


Assuntos
Ressonância de Plasmônio de Superfície , Aflatoxina B1 , Imunoensaio , Micotoxinas , Soroalbumina Bovina
7.
Nanotechnology ; 25(24): 245103, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24872113

RESUMO

We developed Pyrene-Gadolinium (Py-Gd) nanoparticles as pH-sensitive magnetic resonance imaging (MRI) contrast agents capable of showing a high-Mr signal in cancer-specific environments, such as acidic conditions. Py-Gd nanoparticles were prepared by coating Py-Gd, which is a complex of gadolinium with pyrenyl molecules, with pyrenyl polyethyleneglycol PEG using a nano-emulsion method. These particles show better longitudinal relaxation time (T1) MR signals in acidic conditions than they do in neutral conditions. Furthermore, the particles exhibit biocompatibility and MR contrast effects in both in vitro and in vivo studies. From these results, we confirm that Py-Gd nanoparticles have the potential to be applied for accurate cancer diagnosis and therapy.


Assuntos
Meios de Contraste/síntese química , Gadolínio , Imageamento por Ressonância Magnética/instrumentação , Nanopartículas Metálicas , Neoplasias/diagnóstico , Animais , Células 3T3 BALB , Linhagem Celular Tumoral , Materiais Revestidos Biocompatíveis , Gadolínio/química , Humanos , Imageamento por Ressonância Magnética/métodos , Nanopartículas Metálicas/química , Camundongos , Polietilenoglicóis/química , Pirenos/química
8.
Food Chem ; 438: 138043, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-37992606

RESUMO

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.


Assuntos
Listeria monocytogenes , Listeriose , Humanos , Listeria monocytogenes/genética , Listeriose/metabolismo , Listeriose/microbiologia , Proteínas de Fluorescência Verde/genética , Lipossomos/metabolismo , Proteínas Hemolisinas/genética
9.
ACS Omega ; 9(34): 36531-36539, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39220521

RESUMO

Natural polyphenols found in health supplements and drinks have antioxidant and anti-inflammatory properties. In particular, to determine the beneficial qualities of antioxidant drinks and beverages, consumers demand precise quantification of the total amount of polyphenols as on-site detection. Herein, we developed a new concept of portable beads suitable for the field detection available: colorimetric quantification of polyphenols equipped with color converting software applications in a smartphone or tablet PC. The yellowish beads contain ferric ions to react with polyphenol to produce blackish metal-phenolic complexes. It is simple to perform the detection procedure: dipping the beads in the analytical sample and out-taking a photo-converting into RGB color values and quantification of the existed polyphenol. The overall process was completed within 5 min. Compared with the Folin-Ciocalteu assay, which is a representative optical sensor kit for total phenolic content, the bead-based sensor showed a better limit of detection of 0.0415 mM for tannic acid and comparable sensing capability for a polyphenol-containing plant extract and brewed tea. The beads conserved the shape and sensitivity after months of storage or under environmental interference such as a change in the temperature.

10.
Biosens Bioelectron ; 258: 116347, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38723332

RESUMO

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues.


Assuntos
Biomarcadores Tumorais , Técnicas Biossensoriais , Neoplasias da Mama , Aprendizado Profundo , Exossomos , Receptor ErbB-2 , Análise Espectral Raman , Trastuzumab , Trastuzumab/uso terapêutico , Animais , Exossomos/química , Feminino , Camundongos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/urina , Análise Espectral Raman/métodos , Humanos , Biomarcadores Tumorais/urina , Imunoensaio/métodos , Antineoplásicos Imunológicos/uso terapêutico
11.
Talanta ; 275: 126073, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38688085

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) has a high incidence in infectious hospitals and communities, highlighting the need for early on-site detection due to its resistance to methicillin antibiotics. The present study introduces a highly sensitive detection system for mecA, a crucial methicillin marker, utilizing an RCA-based isothermal exponential amplification reaction. The G-quadruplex-based isothermal exponential amplification reaction (GQ-EXPAR) method designs probes to establish G-quadruplex secondary structures incorporating thioflavin T for fluorescence. The system, unlike conventional genetic detection methods, works with portable isothermal PCR devices (isoQuark), facilitating on-site detection. A detection limit of 0.1 fmol was demonstrated using synthetic DNA, and effective detection was proven using thermal lysis. The study also validated the detection of targets swabbed from surfaces within bacterial 3D nanostructures using the GQ-EXPAR method. After applying complementary sequences to the padlock probe for the target, the GQ-EXPAR method can be used on various targets. The developed method could facilitate rapid and accurate diagnostics within MRSA strains.


Assuntos
Quadruplex G , Staphylococcus aureus Resistente à Meticilina , Técnicas de Amplificação de Ácido Nucleico , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Limite de Detecção , Proteínas de Ligação às Penicilinas , Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/análise , Benzotiazóis/química , Humanos
12.
Biosens Bioelectron ; 259: 116375, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38749283

RESUMO

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.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , COVID-19 , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Humanos , Técnicas Biossensoriais/métodos , Aptâmeros de Nucleotídeos/química , COVID-19/virologia , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas do Nucleocapsídeo de Coronavírus/genética , Fosfoproteínas/química , Limite de Detecção , Teste de Ácido Nucleico para COVID-19/métodos , Teste de Ácido Nucleico para COVID-19/instrumentação
13.
ACS Nano ; 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39248519

RESUMO

Molecular-profiling-based cancer diagnosis has significant implications for predicting disease prognosis and selecting targeted therapeutic interventions. The analysis of cancer-derived extracellular vesicles (EVs) provides a noninvasive and sequential method to assess the molecular landscape of cancer. Here, we developed an all-in-one fusogenic nanoreactor (FNR) encapsulating DNA-fueled molecular machines (DMMs) for the rapid and direct detection of EV-associated microRNAs (EV miRNAs) in a single step. This platform was strategically designed to interact selectively with EVs and induce membrane fusion under a specific trigger. After fusion, the DMMs recognized the target miRNA and initiated nonenzymatic signal amplification within a well-defined reaction volume, thus producing an amplified fluorescent signal within 30 min. We used the FNRs to analyze the unique expression levels of three EV miRNAs in various biofluids, including cell culture, urine, and plasma, and obtained an accuracy of 86.7% in the classification of three major breast cancer (BC) cell lines and a diagnostic accuracy of 86.4% in the distinction between patients with cancer and healthy donors. Notably, a linear discriminant analysis revealed that increasing the number of miRNAs from one to three improved the accuracy of BC patient discrimination from 78.8 to 95.4%. Therefore, this all-in-one diagnostic platform performs nondestructive EV processing and signal amplification in one step, providing a straightforward, accurate, and effective individual EV miRNA analysis strategy for personalized BC treatment.

14.
Nanotechnology ; 24(47): 475103, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24192299

RESUMO

Cancer cells can express specific biomarkers, such as cell membrane proteins and signaling factors. Thus, finding biomarkers and delivering diagnostic agents are important in the diagnosis of cancer. In this study, we investigated a biomarker imaging agent for the diagnosis of hepatic cancers. The asialoglycoprotein receptor (ASGPr) was selected as a biomarker for hepatoma cells and the ASGPr-targetable imaging agent bearing a galactosyl group was prepared using manganese ferrite nanoparticles (MFNP) and galactosylgluconic acid. The utility of the ASGPr-targetable imaging agent, galactosylated MFNP (G-MFNP) was assessed by several methods in ASGPr-expressing HepG2 cells as target cells and ASGPr-deficient MCF7 cells. Physical and chemical properties of G-MFNP were examined using Fourier-transform infrared spectroscopy, dynamic light scattering, zeta potential analysis, and transmission electron microscopy. No significant cytotoxicity was observed in either cell line. Targeting ability was assessed using flow cytometry, magnetic resonance imaging, inductively coupled plasma atomic emission spectroscopy, absorbance analysis, dark-field microscopy, Prussian blue staining, and transmission electron microscopy. We demonstrated that G-MFNP target successfully and bind to ASGPr-expressing HepG2 cells specifically. We suggest that these results will be useful in strategies for cancer diagnoses based on magnetic resonance imaging.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , Compostos Férricos/metabolismo , Galactose/metabolismo , Imageamento por Ressonância Magnética , Compostos de Manganês/metabolismo , Nanopartículas/química , Coloides/química , Fluorescência , Glicosilação , Células Hep G2 , Humanos , Células MCF-7 , Nanopartículas/ultraestrutura , Tamanho da Partícula , Espectrofotometria Atômica , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
15.
Biosens Bioelectron ; 241: 115700, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37757509

RESUMO

The simultaneous infection with a tripledemic-simultaneous infection with influenza A pH1N1 virus (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV)-necessitates the development of accurate and fast multiplex diagnostic tests. The coronavirus disease 2019 (COVID-19) pandemic has emphasized the importance of virus detection. Field-effect transistor (FET)-based immuno-biosensors have a short detection time and do not require labeling or polymerase chain reaction. This study demonstrates the rapid, sensitive detection of influenza A pH1N1, SARS-CoV-2, and RSV using a multiplex immunosensor based on a dual-gate oxide semiconductor thin-film transistor (TFT), a type of FET. The dual-gate oxide TFT was modified by adjusting both top and bottom gate insulators to improve capacitive coupling to approximately 120-fold amplification, exhibiting a high pH sensitivity of about 10 V/pH. The dual-gate oxide TFT-based immunosensor detected the target proteins (hemagglutinin (HA) protein of Flu, spike 1 (S1) protein of SARS-CoV-2, and fusion protein of RSV) of each virus, with a limit of detection of approximately 1 fg/mL. Cultured viruses in phosphate-buffered saline or artificial saliva and clinical nasopharynx samples were detected in 1-µL sample volumes within 60 s. This promising diagnosis could be potentially as point-of-care tests to facilitate a prompt response to future pandemics with high sensitivity and multiplexed detection without pretreatment.

16.
Biosens Bioelectron ; 239: 115592, 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37603987

RESUMO

Exosomes are useful for cancer diagnosis and monitoring. However, clinical samples contain impurities that complicate direct analyses of cancer-derived exosomes. Therefore, a microfluidic chip-based magnetically labeled exosome isolation system (MEIS-chip) was developed as a lab-on-a-chip platform for human epidermal growth factor receptor 2 (HER2)-positive cancer diagnosis and monitoring. Various magnetic nanoclusters (MNCs) were synthesized with different degrees of magnetization, and antibodies were introduced to capture HER2-overexpressing and common exosomes using immunoaffinity. MNC-bonded exosomes were separated into different exits according to their magnetization degrees. The MEIS-chip efficiently separated HER2-overexpressing exosomes from common exosomes that did not contain disease-related information. The simultaneous separation of HER2-and non-HER2-overexpressing exosomes provided a means of analyzing high-purity HER2-overexpressing exosomes while minimizing the contribution of non-target exosomes, reducing misdiagnosis risk. Notably, common exosomes served as a negative control for monitoring real-time changes in HER2 expression. These findings support the application of MEIS-chip for cancer diagnosis and treatment monitoring via effective exosome isolation.


Assuntos
Técnicas Biossensoriais , Exossomos , Neoplasias , Humanos , Microfluídica , Neoplasias/diagnóstico , Anticorpos
17.
Nat Commun ; 14(1): 8033, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38052830

RESUMO

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.


Assuntos
Antibacterianos , Ácidos Nucleicos , Humanos , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , beta-Lactamases/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Ácidos Nucleicos/metabolismo , Bactérias/genética , DNA/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana
18.
Biosens Bioelectron ; 219: 114819, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36327561

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as next-generation molecular diagnostics. In CRISPR-based diagnostics, Cas12 and Cas13 proteins have been widely employed to detect DNA and RNA, respectively. Herein, we developed a novel hybrid Cas protein capable of detecting universal nucleic acids (DNA and RNA). The CRISPR/hybrid Cas system simultaneously recognizes both DNA and RNA, enabling the dual detection of pathogenic viruses in a single tube. Using wild-type (WT) and N501Y mutant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as detection models, we successfully detected both virus strains with a detection limit of 10 viral copies per reaction without cross-reactivity. Furthermore, it is demonstrated the detection of WT SARS-CoV-2 and N501Y mutant variants in clinical samples by using the CRISPR/hybrid Cas system. The hybrid Cas protein is expected to be utilized in a molecular diagnostic method for infectious diseases, tissue and liquid biopsies, and other nucleic acid biomarkers.

19.
J Hazard Mater ; 460: 132398, 2023 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-37639787

RESUMO

Over the past few years, infections caused by airborne pathogens have spread worldwide, infecting several people and becoming an increasingly severe threat to public health. Therefore, there is an urgent need for developing airborne pathogen monitoring technology for use in confined environments to enable epidemic prevention. In this study, we designed a colorimetry-based bacterial detection platform that uses a clustered regularly interspaced short palindromic repeat-associated protein 12a system to amplify signals and a urease enzyme to induce color changes. Furthermore, we have developed a smartphone application that can distinguish colors under different illumination conditions based on the HSV model and detect three types of disease-causing bacteria. Even synthetic oligomers of a few picomoles of concentration and genomic DNA of airborne bacteria smaller than several nanograms can be detected with the naked eye and using color analysis systems. Furthermore, in the air capture model system, the bacterial sample generated approximately a 2-fold signal difference compared with that in the control group. This colorimetric detection method can be widely applied for public safety because it is easy to use and does not require complex equipment.


Assuntos
Colorimetria , Smartphone , Humanos , Bactérias/genética , Modelos Biológicos , Saúde Pública
20.
ACS Appl Mater Interfaces ; 15(6): 7759-7766, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36748606

RESUMO

Infectious agents such as viruses pose significant threats to human health, being transmitted via direct contact as well as airborne transmission without direct contact, thus requiring rapid detection to prevent the spread of infectious diseases. In this study, we developed a conductive thread-based immunosensor (CT-IS), a biosensor to easily detect the presence of airborne viruses. CT-IS utilizes an antibody that specifically recognizes the HA protein of the pandemic influenza A (pH1N1) virus, which is incorporated into the conductive thread. The antigen-antibody interaction results in increased strain on the conductive thread in the presence of the pH1N1 virus, resulting in increased electrical resistance of the CT-IS. We evaluated the performance of this sensor using the HA protein and the pH1N1 virus, in addition to samples from patients infected with the pH1N1 virus. We observed a significant change in resistance in the pH1N1-infected patient samples (positive: n = 11, negative: n = 9), whereas negligible change was observed in the control samples (patients not infected with the pH1N1 virus; negative). Hence, the CT-IS is a lightweight fiber-type sensor that can be used as a wearable biosensor by combining it with textiles, to detect the pH1N1 virus in a person's vicinity.


Assuntos
Técnicas Biossensoriais , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Humanos , Influenza Humana/diagnóstico , Imunoensaio , Anticorpos
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