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MicroRNAs (miRNAs) serve as emerging biomarkers for a range of diseases, and their quantitative analysis draws increasing attention. Yet, current invasive methods limit continuous tracking within living cells. To overcome this, a nonenzymatic DNA-based nanoprobe is developed for dynamic, noninvasive miRNA tracking via live-cell imaging. This probe features a unique hairpin DNA structure with five guanines that act as internal quenchers, suppressing fluorescence from an attached fluorophore via photoinduced electron transfer. Target miRNA initiates toehold-mediated strand displacement, restoring, and amplifying the fluorescence signal. Additionally, by introducing a single mismatch to the hairpin DNA, the nanoprobe's sensitivity is significantly enhanced, lowering the detection limit to about 60 pM without compromising specificity. To optimize intracellular delivery for prolonged monitoring, the nanoprobe is encapsulated within multilamellar lipid nanovesicles, fluorescently labeled for dual-wavelength ratiometric analysis. The proposed nanoprobe demonstrates a significant advance in live-cell miRNA detection, promising enhanced in situ analysis for a better understanding of miRNAs' pathophysiological function.
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Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.
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Sistemas CRISPR-Cas , Edição de Genes , Lipossomos , Nanopartículas , Linhagem Celular , Ribonucleoproteínas/genéticaRESUMO
The objective of this study was to evaluate the leachate and textural characteristics of cooked rice, and the correlations between the leachate properties and texture attributes were also investigated. Cooked waxy rice had much higher total solids and amylopectin amount in leachate than the normal and high-amylose rice. For all varieties, the amylopectin chain length of the leachate was similar, excluding Dodam cultivar. The rheological characteristics of the leachate solutions were highly dependent on the amylopectin amount of the leachate. Regarding the textural characteristics, Dodam had the highest hardness and the lowest adhesiveness. The principal component analysis showed substantial differences in leachate and textural characteristics of Korean cooked rice according to its amylose content. The adhesiveness was positively and negatively correlated with amylopectin amount of leachate and the proportion of long amylopectin chains, respectively. These results indicated that the leachate characteristics of cooked rice significantly influenced its textural attributes. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01446-3.
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BACKGROUND: Physically modified starches can be classified as natural ingredients on food labels and clean label products. Thus, the market demand for physically modified starch is increasing. Potato, tapioca and corn starches were physically modified by mild heat treatment in an alcoholic solution to enhance their gelling property and freeze-thaw stability. RESULTS: During mild heating of starch suspension (40% w/w) in 10% ethanol solution at the onset gelatinization temperature, granular swelling of starch occurred, followed by amylose leaching with medication of the surface structure of the starch granules. All treated starches exhibited increased gelatinization and pasting temperatures and decreased breakdown for pasting as a result of improved stability against shear and heat. The treated starches had higher hardness, cohesiveness and springiness of gel than the respective native starches, and these gel properties were more pronounced in potato starch than in tapioca and corn starches. The treated starches showed substantially reduced gel syneresis during freeze-thawing. CONCLUSION: Physical modification of starch by mild heat treatment in an alcoholic solution substantially improved its gelation ability and freeze-thaw stability. © 2024 Society of Chemical Industry.
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Etanol , Congelamento , Géis , Temperatura Alta , Manihot , Solanum tuberosum , Amido , Zea mays , Amido/química , Solanum tuberosum/química , Zea mays/química , Géis/química , Etanol/química , Manihot/química , Manipulação de Alimentos/métodos , Tubérculos/química , Amilose/química , Amilose/análiseRESUMO
The CRISPR/Cas system has been introduced as an innovative tool for therapy, however achieving specific delivery to the target has been a major challenge. Here, an antibody-CRISPR/Cas conjugate platform that enables specific delivery and target gene editing in HER2-positive cancer is introduced. The CRISPR/Cas system by replacing specific residues of Cas9 with an unnatural amino acid is engineered, that can be complexed with a nanocarrier and bioorthogonally functionalized with a monoclonal antibody targeting HER2. The resultant antibody-conjugated CRISPR/Cas nanocomplexes can be specifically delivered and induce gene editing in HER2-positive cancer cells in vitro. It is demonstrated that the in vivo delivery of the antibody-CRISPR/Cas nanocomplexes can effectively disrupt the plk1 gene in HER2-positive ovarian cancer, resulting in substantial suppression of tumor growth. The current study presents a useful therapeutic platform for antibody-mediated delivery of CRISPR/Cas for the treatment of various cancers and genetic diseases.
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Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Humanos , Camundongos , Animais , Feminino , Linhagem Celular Tumoral , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Anticorpos Monoclonais/genética , Neoplasias/terapia , Neoplasias/genética , Receptor ErbB-2/genéticaRESUMO
In this study, malt was produced in pilot-scale facilities and conditioned using three barley (Hordeum vulgare L.) cultivars in South Korea (Heugho, Hopum, and Kwangmaeg). Quality and starch characteristics were compared. The starch content was considerably reduced in all malts. Coleoptile elongation was higher in Heugho (HHM; 85.7% ± 12.6%) and Hopum (HPM; 83.9% ± 10.7%) than in Kwangmaeg (KMM; 78.1% ± 9.9%) malt. Malt yield ranged from 81.8 to 84.9%, with no significant difference. All samples presented type A crystallinity, and granules showed discoid shapes. After malting, the mono- and di-saccharide contents (not including sucrose) were increased. The fermentable sugar level was the highest in HHM, whereas non-fermentable sugar was the highest in KMM. These results suggest that HPM enables efficient scarification based on the rapid degradation of starch, while Heugho barley and HHM have a high potential for beer and malt production, respectively. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01419-6.
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The consumption of take-out food has increased worldwide; consequently, people are increasingly being exposed to chemicals from food containers. However, research on the migration of metals from containers to food is limited, and therefore, information required to determine the health risks is lacking. Herein, the amount of transfer of nine metals and metalloids (Pb, Sb, Cd, Ge, Co, Mn, Sn, As, and Hg) from food containers to food in South Korea was assessed from take-out food containers classified into paper and plastic container groups. The sample containers were eluted over time by either warming with 4% acetic acid at 70 °C or cooling with 4% acetic acid at 100 °C /deionized water at 25 °C. It was analyzed using an inductively coupled plasma mass spectrometer and a direct mercury analyzer. The reliability of the quantitative results was verified by calculating the linearity, limit of detection, and limit of quantification. We found that the amount of metals and metalloids (Pb, Sb, Cd, and Co) eluting over time was highly significant in the plastic group. Regardless of the food simulant and elution time, the amount of Sb transferred from the food containers to food was substantially higher in the plastic (average concentration: 0.488-1.194 µg/L) than in the paper group (average concentration: 0.001-0.03 µg/L). Fortunately, all food containers were distributed at levels safe for human health (hazard index: 0.000-64.756%). However, caution is needed when warm food is added to food containers. Overall, our results provide baseline data for the management and use of take-out containers.
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Mercúrio , Metaloides , Metais Pesados , Humanos , Metaloides/análise , Cádmio/análise , Embalagem de Alimentos , Chumbo , Reprodutibilidade dos Testes , Metais Pesados/análise , Mercúrio/análise , República da Coreia , Acetatos , Medição de Risco , Monitoramento Ambiental/métodosRESUMO
Rapid and accurate diagnosis is crucial for managing the global health threat posed by multidrug-resistant bacterial infections; however, current methods have limitations in either being time-consuming, labor-intensive, or requiring instruments with high costs. Addressing these challenges, we introduce a wireless electrochemical sensor integrating the CRISPR/Cas system with electroconductive polymer dot (PD) nanoparticles to rapidly detect bacterial pathogens from human sputum. To enhance the electroconductive properties, we synthesized copper-ion-immobilized PD (PD-Cu), followed by conjugation of the deactivated Cas9 protein (dCas9) onto PD-Cu-coated Si electrodes to generate the dCas9-PD-Cu sensor. The dCas9-PD-Cu sensor integrated with isothermal amplification can specifically detect target nucleic acids of multidrug-resistant bacteria, such as the antibiotic resistance genes kpc-2 and mecA. The dCas9-PD-Cu sensor exhibits high sensitivity, allowing for the detection of â¼54 femtograms of target nucleic acids, based on measuring the changes in resistivity of the Si electrodes through target capture by dCas9. Furthermore, a wireless sensing platform of the dCas9-PD-Cu sensor was established using a Bluetooth module and a microcontroller unit for detection using a smartphone. We demonstrate the feasibility of the platform in diagnosing multidrug-resistant bacterial pneumonia in patients' sputum samples, achieving 92% accuracy. The current study presents a versatile biosensor platform that can overcome the limitations of conventional diagnostics in the clinic.
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Ácidos Nucleicos , Pneumonia Bacteriana , Humanos , Polímeros , Cobre/química , Resistência a Múltiplos MedicamentosRESUMO
Simple diagnostic tests for nucleic acid targets can provide great advantages for applications such as rapid pathogen detection. Here, we developed a membrane assay for multiplexed detection of nucleic acid targets based on the visualization of two-dimensional fluorescent ring patterns. A droplet of the assay solution is applied to a cellulose nitrate membrane, and upon radial chromatographic flow and evaporation of the solvent, fluorescent patterns appear under UV irradiation. The target nucleic acid is isothermally amplified and is immediately hybridized with fluorescent oligonucleotide probes in a one-pot reaction. We established the fluorescent ring assay integrated with isothermal amplification (iFluor-RFA = isothermal fluorescent ring-based radial flow assay), and feasibility was tested using nucleic acid targets of the receptor binding domain (RBD) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2. We demonstrate that the iFluor-RFA method is capable of specific and sensitive detection in the subpicomole range, as well as multiplexed detection even in complex solutions. Furthermore, we applied deep learning analysis of the fluorescence images, showing that patterns could be classified as positive or negative and that quantitative amounts of the target could be predicted. The current technique, which is a membrane pattern-based nucleic acid assay combined with deep learning analysis, provides a novel approach in diagnostic platform development that can be versatilely applied for the rapid detection of infectious pathogens.
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Aprendizado Profundo , Ácidos Nucleicos , Ácidos Nucleicos/análise , Sondas de Oligonucleotídeos , SARS-CoV-2/genética , Corantes Fluorescentes/química , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
BACKGROUND: Abdominal pain, which is a common cause of children presenting to the paediatric emergency department (PED), is often evaluated by ultrasonography (US). However, uncertainty in US reports may necessitate additional imaging. OBJECTIVE: In this study, we evaluated factors contributing to uncertainty in paediatric abdominal US reports in the PED. MATERIALS AND METHODS: This retrospective cohort study included children younger than 18 years of age who underwent abdominal US in the PED of the study hospital between January 2017 and December 2019. After exclusion, the researchers manually reviewed and classified all US reports as 'certain' or 'uncertain'. Univariate and multivariate logistic regression analyses were performed to identify the factors contributing to uncertain reports. RESULTS: In total, 1006 patients were included in the final analysis., 796 patients were tagged as having certain reports, and 210 as having uncertain reports. Children with uncertain reports had a significantly higher rate of undergoing an additional computed tomography (CT) scan (31.0% vs. 2.5%, p < 0.001) and a longer PED median length of stay (321.0 (Interquartile range (IQR); 211.3-441.5) minutes vs. 284.5 (IQR; 191.8-439.5) minutes, p = 0.042). After logistic regression, US performed by a radiology resident (odds ratio, 5.01; 95% confidence interval, 3.63-7.15) was the most significant factor contributing to uncertainty in paediatric abdominal US reports followed by obesity and age. CONCLUSION: Several factors contribute to uncertainty in paediatric abdominal US reports. Uncertain radiological reports increase the likelihood of additional CT scans. Measures to improve the clarity of radiological reports must be considered to improve the quality of care for children visiting the PED.
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Dor Abdominal , Serviço Hospitalar de Emergência , Criança , Humanos , Estudos Retrospectivos , Incerteza , Dor Abdominal/diagnóstico por imagem , UltrassonografiaRESUMO
Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.
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Sistemas CRISPR-Cas , Técnicas de Transferência de Genes , Humanos , Sistemas CRISPR-Cas/genética , Preparações Farmacêuticas , Nanomedicina , Edição de GenesRESUMO
This study aimed to explore suitable processing materials for rice beer (RB) production by analyzing the starch structure of the raw materials utilized for brewing beer and the quality characteristics of RB. We used malt, employing the Heugho cultivar as the main ingredient, and produced beer containing 30% rice. The regular amylose-containing cultivars Samgwang (SA) and Hangaru (HA) and the high-amylose-containing cultivar Dodamssal (DO) were used as adjuncts. Distribution of the short molecular chains of the starch amylopectin was the highest for SA and malt at 29.3% and 27.1%, respectively. Glucose content was the highest in the wort prepared with 100% malt and 30% SA + 70% malt. The alcohol content in SA RB and HA RB was higher than that in beer prepared with 100% malt. DO RB had the least bitterness and volatile components, such as acetaldehyde and ethyl acetate. The three rice cultivars tested in this study are suitable as starch adjuncts for RB production. The characteristics of RBs varied depending on the molecular structure of the ingredients, irrespective of their amylose contents. SA could be considered a craft beer with quality characteristics and rich flavor components, similar to 100% malt beer, compared to other RBs.
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Rice is a major source of carbohydrates. Resistant starch (RS) is digested in the human small intestine but fermented in the large intestine. This study investigated the effect of consuming heat-treated and powdered brown rice cultivars 'Dodamssal' (HBD) and 'Ilmi' (HBI), with relatively high and less than 1% RS content, respectively, on the regulation of glucose metabolism in humans. Clinical trial meals were prepared by adding ~80% HBI or HBD powder to HBI and HBD meals, respectively. There was no statistical difference for protein, dietary fiber, and carbohydrate content, but the median particle diameter was significantly lower in HBI meals than in HBD meals. The RS content of HBD meals was 11.4 ± 0.1%, and the HBD meals also exhibited a low expected glycemic index. In a human clinical trial enrolling 36 obese participants, the homeostasis model assessment for insulin resistance decreased by 0.05 ± 0.14% and 1.5 ± 1.40% after 2 weeks (p = 0.021) in participants in the HBI and HBD groups, respectively. The advanced glycation end-product increased by 0.14 ± 0.18% in the HBI group and decreased by 0.06 ± 0.14% in the HBD group (p = 0.003). In conclusion, RS supplementation for 2 weeks appears to have a beneficial effect on glycemic control in obese participants.
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Oryza , Amido , Humanos , Amido/metabolismo , Amido Resistente/metabolismo , Oryza/metabolismo , Temperatura Alta , Carboidratos , Obesidade , Glucose/metabolismo , Glicemia/metabolismo , Insulina/metabolismoRESUMO
The challenge in effective delivery of mRNA has been a major hurdle in their development as therapeutics. Herein, we present that the incorporation of cationic nanogels as the condensing material for mRNA into liposomes enables stable and enhanced mRNA delivery to cells in vitro. We prepared dextran-based nanogel particles, which were surface functionalized with oligoarginine peptide (DNPR9) and complexed with mRNA for incorporation into liposomes (LipoDNPR9). The use of DNPR9 with the liposomes resulted in enhanced internalization, as well as a 4-fold increase in transfection of luciferase mRNA when treated with A549 cells in vitro, compared to control liposomes. The enhancement in transfection efficiency was also observed in various cell lines while causing low cytotoxicity. The versatility of the strategy was also investigated by applying DNPR9 for mRNA condensation to ionizable lipid particles, which resulted in an â¼55% increase in transfection. The current development based on nanogel-incorporated liposomes introduces an effective platform for mRNA delivery, while the condensation strategy using DNPR9 can be widely applied for various lipid-based formulations to enhance their efficacy.
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Lipídeos , Lipossomos , Nanogéis , RNA Mensageiro/genética , TransfecçãoRESUMO
Using improper wound care materials may cause impaired wound healing, which can involve scar formation and infection. Herein, we propose a facile method to fabricate a cell-alignment scaffold, which can effectively enhance cell growth and migration, leading to the reproduction of cellular arrangements and restoration of tissues. The principle is scratching a diamond lapping film that gives uniaxial nanotopography on substrates. Cells are seeded to follow the geometric cue via contact guidance, resulting in highly oriented cell alignment. Remarkable biocompatibility is also demonstrated by the high cell viability on various substrates. In vivo studies in a wound healing model in mice show that the scratched film supports directed cell guidance on the nanostructure, with significantly reduced wound areas and inhibition of excessive collagen deposition. Rapid recovery of the epidermis and dermis is also shown by histological analyses, suggesting the potential application of the scratching technique as an advanced wound dressing material for effective tissue regeneration.
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Colágeno , Cicatrização , Camundongos , Animais , Colágeno/química , Proliferação de Células , BandagensRESUMO
Purpose: This study aimed to investigate radiomics analysis of ultrasonographic images to develop a potential biomarker for predicting lymph node metastasis in papillary thyroid carcinoma (PTC) patients. Materials and Methods: This study included 431 PTC patients from August 2013 to May 2014 and classified them into the training and validation sets. A total of 730 radiomics features, including texture matrices of gray-level co-occurrence matrix and gray-level run-length matrix and single-level discrete two-dimensional wavelet transform and other functions, were obtained. The least absolute shrinkage and selection operator method was used for selecting the most predictive features in the training data set. Results: Lymph node metastasis was associated with the radiomics score (p < 0.001). It was also associated with other clinical variables such as young age (p = 0.007) and large tumor size (p = 0.007). The area under the receiver operating characteristic curve was 0.687 (95% confidence interval: 0.616-0.759) for the training set and 0.650 (95% confidence interval: 0.575-0.726) for the validation set. Conclusion: This study showed the potential of ultrasonography-based radiomics to predict cervical lymph node metastasis in patients with PTC; thus, ultrasonography-based radiomics can act as a biomarker for PTC.
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This study investigated leachate and morphological properties of electric-cooked rice (ECR), electric pressure-cooked rice (EPCR), and instant cooked rice (ICR) to explore the effects of cooking methods on eating quality of cooked rice. The leachate was obtained by rinsing cooked rice with warm water. EPCR had the highest amounts of total solid and amylopectin in the leachate and the highest contents of surface and bound lipid. The amylopectin branch chain length of leachate was not significantly different among rice samples. EPCR leachate solution showed the highest apparent viscosity and the greatest decline with increasing shear rate due to high amount of amylopectin. In morphological characteristics, degrees for disruption of the starch structure and compression of protein present in rice kernel were largest in EPCR. Textural hardness of ICR was much lower than that of ECR or EPCR. EPCR had the highest glossiness, stickiness, moistness, and overall acceptability scores. Principal component analysis score plot showed significant differences in leachate and textural characteristics of cooked rice according to cooking methods.
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Oryza , Amido , Amilopectina , Receptor de Proteína C Endotelial , CulináriaRESUMO
The objective of the present study is to enhance the resistant starch (RS) content of high amylose rice starch with heat-moisture treatment (HMT) for industrial application. The optimized HMT condition for achieving the highest RS content established using response surface methodology (RSM) was a temperature of 100 °C, moisture content of 24.2%, and a time of 11.5 h. Upon HMT, the RS content increased from 32.1% for native starch to 46.4% in HMT starch with optimized condition. HMT of the starches reduced the solubility and swelling power. The surface of HMT starch granules was more irregular than native starch. The X-ray diffraction (XRD) peak intensity at 2θ = 5° was greatly reduced by HMT, and the peaks at 22.7° and 24.2° were merged. HMT increased the gelatinization temperature and reduced the gelatinization enthalpy. HMT provides a method for the production of high-yield RS2 with high amylose rice starch in industrial application.
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Amilose , Oryza , Temperatura Alta , Amido Resistente , Amido , Água , Difração de Raios XRESUMO
We introduce a lateral flow assay (LFA) integrated with a modified isothermal nucleic acid amplification procedure for rapid and simple genetic testing. Padlock probes specific for the target DNA were designed for ligation, followed by rolling circle amplification (RCA) using capture ligand-modified oligonucleotides as primers. After hybridization with detection linker probes, the amplified target DNA is flowed through an LFA membrane strip for binding of gold nanoparticles as the substrate for colorimetric detection. We established and validated the "RCA-LFA" method for detection of mecA, the antibiotic resistance gene for methicillin-resistant Staphylococcus aureus (MRSA). The assay was optimized using various concentrations of primers and probes for RCA and LFA, respectively. The sensitivity was determined by performing RCA-LFA using various amounts of mecA target DNA, showing a detection limit of ~ 1.3 fmol. The specificity of the assay was examined using target DNAs for other resistance genes as the controls, which demonstrated positive detection signals only for mecA DNA, when added either individually or in combinations with the control targets. Furthermore, applying the RCA-LFA method using specifically designed probes for RNA-dependent RNA polymerase (RdRp) and receptor binding domain (RBD) gene for SARS-CoV-2, which demonstrated feasibility of the method for viral gene targets. The current method suggests a useful platform which can be universally applied for various nucleic acid targets, allowing rapid and sensitive diagnosis at point-of-care. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00080-1.
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The healthcare industry is in dire need of rapid microbial identification techniques for treating microbial infections. Microbial infections are a major healthcare issue worldwide, as these widespread diseases often develop into deadly symptoms. While studies have shown that an early appropriate antibiotic treatment significantly reduces the mortality of an infection, this effective treatment is difficult to practice. The main obstacle to early appropriate antibiotic treatments is the long turnaround time of the routine microbial identification, which includes time-consuming sample growth. Here, we propose a microscopy-based framework that identifies the pathogen from single to few cells. Our framework obtains and exploits the morphology of the limited sample by incorporating three-dimensional quantitative phase imaging and an artificial neural network. We demonstrate the identification of 19 bacterial species that cause bloodstream infections, achieving an accuracy of 82.5% from an individual bacterial cell or cluster. This performance, comparable to that of the gold standard mass spectroscopy under a sufficient amount of sample, underpins the effectiveness of our framework in clinical applications. Furthermore, our accuracy increases with multiple measurements, reaching 99.9% with seven different measurements of cells or clusters. We believe that our framework can serve as a beneficial advisory tool for clinicians during the initial treatment of infections.