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1.
Crit Rev Biotechnol ; : 1-17, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38503543

RESUMO

As an important cell factory, industrial yeast has been widely used for the production of compounds ranging from bulk chemicals to complex natural products. However, various adverse conditions including toxic products, extreme pH, and hyperosmosis etc., severely restrict microbial growth and metabolic performance, limiting the fermentation efficiency and diminishing its competitiveness. Therefore, enhancing the tolerance and robustness of yeasts is critical to ensure reliable and sustainable production of metabolites in complex industrial production processes. In this review, we provide a comprehensive review of various strategies for improving the tolerance of yeast cells, including random mutagenesis, system metabolic engineering, and material-mediated immobilization cell technology. It is expected that this review will provide a new perspective to realize the response and intelligent regulation of yeast cells to environmental stresses.

2.
Anal Chem ; 95(2): 1541-1548, 2023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36595491

RESUMO

Multiplexed protein detection is critical for improving the drug and biomarker screening efficiency. Here, we show that multiplexed protein detection and parallel protein interaction analysis can be realized by evanescent scattering microscopy (ESM). ESM enables binding kinetics measurement with label-free digital single-molecule counting. We implemented an automatic single-molecule counting strategy with high temporal resolution to precisely determine the binding time, which improves the counting efficiency and accuracy. We show that digital single-molecule counting can recognize proteins with different molecular weights, thus making it possible to monitor the protein binding processes in the solution by real-time tracking of the numbers of free and bound proteins landing on the sensor surface. Furthermore, we show that this strategy can simultaneously analyze the kinetics of two different protein interaction processes on the surface and in the solution. This work may pave a way to investigate complicated protein interactions, such as the competition of biomarker-antibody binding in biofluids with biomarker-protein binding on the cellular membrane.


Assuntos
Microscopia , Proteínas , Proteínas/metabolismo , Membrana Celular/metabolismo , Nanotecnologia , Biomarcadores/metabolismo , Cinética , Ligação Proteica
3.
Nat Methods ; 17(10): 1010-1017, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32958922

RESUMO

Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.


Assuntos
Proteínas/química , Imagem Individual de Molécula , Humanos , Imunoglobulina A/química , Imunoglobulina A/metabolismo , Imunoglobulina M/química , Imunoglobulina M/metabolismo , Cinética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Ressonância de Plasmônio de Superfície
4.
Anal Chem ; 94(42): 14503-14508, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36223252

RESUMO

Plasmonic absorption of light can create significant local heat and has become a promising tool for rapid temperature regulation in diverse fields, from biomedical technology to optoelectronics. Current plasmonic heating usually relies on specially designed nanomaterials randomly distributed in the space and barely provides uniform temperature regulation in a wide field. Herein, we report a rapid temperature regulation strategy on a plain gold-coated glass slip using a plasmonic scattering microscopy, which can be referred to as wide-field plasmonic thermal microscopy (W-PTM). We calibrated the W-PTM by monitoring the phase transition of the temperature-sensitive polymer solutions, showing that it can provide a temperature regulation range of 33-80 °C. Moreover, the W-PTM provides imaging capability, thus allowing the statistical analysis of the phase-transitioned polymeric nanoparticles. Finally, we demonstrated that W-PTM can be used for noninvasive and local regulation of the transient receptor potential vanilloid 1 (TRPV1) ion channels in the living cells, which can be monitored by simultaneous fluorescence imaging of the calcium influx. With the nondestructive local temperature-regulating and concurrent fluorescence imaging capability, we anticipate that W-PTM can be a powerful tool to study cellular activities associated with cellular membrane temperature changes.


Assuntos
Antineoplásicos , Cálcio , Temperatura , Cálcio/metabolismo , Microscopia , Ouro , Temperatura Alta , Polímeros , Canais Iônicos , Canais de Cátion TRPV/fisiologia
5.
Anal Chem ; 94(30): 10781-10787, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35852494

RESUMO

Single-molecule detection can push beyond ensemble averages and reveal the statistical distributions of molecular properties. Measuring the binding kinetics of single proteins also represents one of the critical and challenging tasks in protein analysis. Here, we report total internal reflection-based evanescent scattering microscopy with label-free single-protein detection capability. Total internal reflection is employed to excite the evanescent field to enhance light-analyte interaction and reduce environmental noise. As a result, the system provides wide-field imaging capability and allows excitation and observation using one objective. In addition, this system quantifies protein binding kinetics by simultaneously counting the binding of individual molecules and recording their binding sites with nanometer precision, providing a digital method to measure binding kinetics with high spatiotemporal resolution. This approach does not employ specially designed microspheres or nanomaterials and may pave a way for label-free single-protein analysis in conventional microscopy.


Assuntos
Nanoestruturas , Microscopia de Fluorescência/métodos
6.
Sensors (Basel) ; 22(18)2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-36146345

RESUMO

Owing to the widespread use of GPS-enabled devices, sensing road information from vehicle trajectories is becoming an attractive method for road map construction and update. Although the detection of intersections is critical for generating road networks, it is still a challenging task. Traditional approaches detect intersections by identifying turning points based on the heading changes. As the intersections vary greatly in pattern and size, the appropriate threshold for heading change varies from area to area, which leads to the difficulty of accurate detection. To overcome this shortcoming, we propose a deep learning-based approach to detect turns and generate intersections. First, we convert each trajectory into a feature sequence that stores multiple motion attributes of the vehicle along the trajectory. Next, a supervised method uses these feature sequences and labeled trajectories to train a long short-term memory (LSTM) model that detects turning trajectory segments (TTSs), each of which indicates a turn occurring at an intersection. Finally, the detected TTSs are clustered to obtain the intersection coverages and internal structures. The proposed approach was tested using vehicle trajectories collected in Wuhan, China. The intersection detection precision and recall were 94.0% and 91.9% in a central urban region and 94.1% and 86.7% in a semi-urban region, respectively, which were significantly higher than those of the previously established local G* statistic-based approaches. In addition to the applications for road map development, the newly developed approach may have broad implications for the analysis of spatiotemporal trajectory data.


Assuntos
Acidentes de Trânsito , Condução de Veículo , China , Cidades , Coleta de Dados , Memória de Curto Prazo
7.
Angew Chem Int Ed Engl ; 61(42): e202209469, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-35922374

RESUMO

Surface plasmon resonance microscopy (SPRM) is an excellent platform for in situ studying cell-substrate interactions. However, SPRM suffers from poor spatial resolution and small field of view. Herein, we demonstrate plasmonic scattering microscopy (PSM) by adding a dry objective on a popular prism-coupled surface plasmon resonance (SPR) system. PSM not only retains SPRM's high sensitivity and real-time analysis capability, but also provides ≈7 times higher spatial resolution and ≈70 times larger field of view than the typical SPRM, thus providing more details about membrane protein response to ligand binding on over 100 cells simultaneously. In addition, PSM allows quantifying the target movements in the axial direction with a high spatial resolution, thus allowing mapping adhesion spring constants for quantitatively describing the mechanical properties of the cell-substrate contacts. This work may offer a powerful and cost-effective strategy for upgrading current SPR products.


Assuntos
Proteínas de Membrana , Microscopia , Cinética , Ligantes , Ligação Proteica , Ressonância de Plasmônio de Superfície
8.
Anal Chem ; 92(8): 5904-5909, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32216299

RESUMO

Charge is a fundamental property of a molecule, and precisely measuring it enables detection of the molecule and helps understand various chemical processes involving charge. Here we show a method to measure the charge of a single nanoparticle and binding of charged molecules to the nanoparticle using a conventional bright field optical microscope. The nanoparticle is tethered to an indium tin oxide surface with a polymer and driven into oscillation with an alternating electric field, which produces scattered light captured by a camera. The weak scattered light is separated from the intense bright field background using a Fourier transform filter, and the image contrast change provides the effective charge of the nanoparticle with precision of a few electron charges or less. This method allows us to detect DNA binding to the nanoparticles, demonstrating a simple method to detect and study molecules with a conventional optical microscope.

9.
Biomed Microdevices ; 19(2): 33, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28429183

RESUMO

Okadaic acid (OA) is a marine toxin ingested by shellfish. In this work, a simple, sensitive and label-free gap-based electrical competitive bioassay has been developed for this biotoxin detection. The gap-electrical biosensor is constructed by modifying interdigitated microelectrodes with gold nanoparticles (AuNPs) and using the self-catalytic growth of AuNPs as conductive bridges. In this development, the AuNPs growth is realized in the solution of glucose and chloroauric acid, with glucose oxidation used as the catalysis for growth of the AuNPs. The catalytic reaction product H2O2 in turn reduces chloroauric acid to make the AuNPs grow. The conductance signal amplification is directly determined by the growth efficiency of AuNPs and closely related to the catalytic activity of AuNPs upon their interaction with OA molecule and OA aptamer. In the absence of OA molecule, the OA aptamer can absorb onto the surfaces of AuNPs due to electrostatic interaction, and the catalytically active sites of AuNPs are fully blocked. Thus the AuNPs growth would not happen. In contrast, the presence of OA molecule can hinder the interaction of OA aptamer and AuNPs. Then the AuNPs sites are exposed and the catalytic growth induces the conductance signal change. The results demonstrated that developed biosensor was able to specifically respond to OA ranging from 5 ppb to 80 ppb, providing limit of detection of 1 ppb. The strategy is confirmed to be effective for OA detection, which indicates the label-free OA biosensor has great potential to offer promising alternatives to the traditional analytical and immunological methods for OA detection.


Assuntos
Técnicas Biossensoriais/instrumentação , Condutividade Elétrica , Ouro/química , Nanopartículas Metálicas/química , Ácido Okadáico/análise , Glucose/química , Oxirredução , Silanos/química
10.
Biosensors (Basel) ; 14(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38391999

RESUMO

The detection and analysis of small molecules, typically defined as molecules under 1000 Da, is of growing interest ranging from the development of small-molecule drugs and inhibitors to the sensing of toxins and biomarkers. However, due to challenges such as their small size and low mass, many biosensing technologies struggle to have the sensitivity and selectivity for the detection of small molecules. Notably, their small size limits the usage of labeled techniques that can change the properties of small-molecule analytes. Furthermore, the capability of real-time detection is highly desired for small-molecule biosensors' application in diagnostics or screening. This review highlights recent advances in label-free real-time biosensing technologies utilizing different types of transducers to meet the growing demand for small-molecule detection.


Assuntos
Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Nanotecnologia , Tecnologia , Biomarcadores , Transdutores
11.
Free Radic Biol Med ; 210: 212-220, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38036070

RESUMO

QSOX1 is a sulfhydryl oxidase that has been identified as a potential biomarker in multiple cancer types as well as acute decompensated heart failure. Three anti-QSOX1 monoclonal antibodies (mAbs) were generated: 2F1, 3A10, and 56-3. MAbs 2F1 and 3A10 were generated against the short isoform of recombinant QSOX1 (rQSOX1-S), and mAb 56-3 was generated against a peptide (NEQEQPLGQWHLS) from the long isoform of QSOX1 (QSOX1-L). Using these mAbs, tandem antigen capture ELISAs were developed to quantify both short and long isoforms of QSOX1 (Total QSOX1 ELISA) and QSOX1-L (QSOX1-L ELISA) in serum and plasma samples. The Total QSOX1 ELISA pairs mAbs 2F1 and 3A10 and has a limit of detection of 109.5 pM, while the QSOX1-L ELISA pairs mAbs 2F1 and 56-3 and has a limit of detection of 10 pM. The levels of total QSOX1 and QSOX1-L were measured in a cohort of paired sera and plasma from 61 donors ≥40 years old and 15 donors <40 years old. No difference in QSOX1 levels was detected between QSOX1-L and QSOX1-S in serum, but the mean concentration of QSOX1-L was found to be 3.21 nM in serum and 5.63 nM in plasma (**p = 0.006). Our tandem ELISAs demonstrate the wide range of concentrations of QSOX1-L and QSOX1-S among individual serum and plasma samples. Since the epitope of mAb 2F1 was mapped to the first CxxC motif at residues C70 and C73 and mAb 56-3 was generated against NEQEQPLGQWHLS in QSOX1-L, our findings support previous research which suggested that QSOX1-L is secreted from cells despite a putative transmembrane domain. The ELISAs reported here may be a useful tool for investigating QSOX1 isoforms as potential biomarkers in cancer and/or heart failure.


Assuntos
Insuficiência Cardíaca , Neoplasias , Humanos , Adulto , Isoformas de Proteínas , Anticorpos Monoclonais/química , Ensaio de Imunoadsorção Enzimática , Oxirredutases atuantes sobre Doadores de Grupo Enxofre
12.
Tuberculosis (Edinb) ; 148: 102531, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38885567

RESUMO

The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrAMtb, dosRMtb, and cydAMtb genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrAMtb bound DNA with greatest affinity. Collectively, wild-type PrrAMtb recombinant protein displayed the highest binding affinity to the dosRMtb promoter (KD 3.46 ± 2.09 nM), followed by the prrAMtb promoter (KD 9.00 ± 2.66 nM). To establish PrrAMtb regulatory activity, we constructed M. smegmatis ΔprrABMsmeg::prrAMtb strains with each of the PrrAMtb variants and extrachromosomal prrAMtb, dosRMtb, and cydAMtb promoter-mCherry reporter fusions. Our findings showed that PrrAMtb is autoregulatory and induces dosRMtb expression only during in vitro, hypoxic growth. Combined expression of prrABMtb in M. smegmatis ΔprrAB significantly induced cydAMtb promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias , Proteínas de Ligação a DNA , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis , Regiões Promotoras Genéticas , Ligação Proteica , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Adaptação Fisiológica/genética , Fosforilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Mutação , Anaerobiose
13.
Eur J Pharmacol ; 982: 176951, 2024 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-39214272

RESUMO

This study aims to identify potential targets and regulatory mechanisms of Astragaloside Ⅳ (AS-Ⅳ) in treating intervertebral disc degeneration (IDD) through network pharmacology analysis with experimental validation. Lumbar spine instability (LSI) mouse models were first established and treated with AS-Ⅳ. Micro-CT, safranin O-fast green staining, IDD score, RT-PCR and immunohistochemistry staining were employed to demonstrate the effect of AS-Ⅳ. Network pharmacology was used to predict the signaling pathways and potential targets of AS-Ⅳ in treating IDD. RT-PCR and immunohistochemistry staining were used to elucidate and validate the mechanism of AS-Ⅳ in vivo. Animal experiments showed that AS-Ⅳ maintained disc height and volume, improved matrix metabolism in LSI mice, and restored Col2α1, ADAMTS-5, Aggrecan, and MMP-13 expression in degenerated discs. Network pharmacology analysis identified 32 cross-targets between AS-Ⅳ and IDD, and PPI network analysis filtered out 11 core genes, including ALB, MAPK1, MAPK14 (p38 MAPK), EGFR, TGFBR1, MAPK8, MMP3, ANXA5, ESR1, CASP3, and IGF1. Enrichment analysis revealed that 7 of the 11 core target genes enriched in the MAPK signaling pathway, and AS-Ⅳ exhibited stable binding to them according to molecular docking results. Experimental validation indicated that AS-Ⅳ reversed mRNA levels of 7 core targets in degenerated disc tissues in LSI mice. Immunohistochemistry staining further revealed that AS-Ⅳ treatment mainly depressed IDD-elevated protein levels of EGFR, p38 MAPK and CASP3 in the annulus fibrosus. This study elucidates that AS-Ⅳ alleviates lumbar spine instability-induced IDD in mice, suggesting the mechanism may involve inhibition of the EGFR/MAPK signaling pathway.


Assuntos
Degeneração do Disco Intervertebral , Farmacologia em Rede , Saponinas , Triterpenos , Animais , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Saponinas/farmacologia , Saponinas/uso terapêutico , Triterpenos/farmacologia , Triterpenos/uso terapêutico , Camundongos , Masculino , Modelos Animais de Doenças , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Vértebras Lombares/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia
14.
J Phys Chem B ; 127(46): 9943-9953, 2023 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-37963180

RESUMO

Study interaction between ligands and protein receptors is a key step for biomarker research and drug discovery. In situ measurement of cell surface membrane protein binding on whole cells eliminates the cost and pitfalls associated with membrane protein purification. Ligand binding to membrane protein was recently found to induce nanometer-scale cell membrane deformations, which can be monitored with real-time optical imaging to quantify ligand/protein binding kinetics. However, the insight into this phenomenon has still not been fully understood. We hypothesize that ligand binding can change membrane stiffness, which induces membrane deformation. To investigate this, cell height and membrane stiffness changes upon ligand binding are measured using atomic force microscopy (AFM). Wheat germ agglutinin (WGA) is used as a model ligand that binds to the cell surface glycoprotein. The changes in cell membrane stiffness and cell height upon ligand bindings are determined for three different cell lines (human A431, HeLa, and rat RBL-2H3) on two different substrates. AFM results show that cells become stiffer with increased height after WGA modification for all cases studied. The increase in cell membrane stiffness is further confirmed by plasmonic scattering microscopy, which shows an increased cell spring constant upon WGA binding. Therefore, this study provides direct experimental evidence that the membrane stiffness changes are directly correlated with ligand binding-induced cell membrane deformation.


Assuntos
Proteínas de Membrana , Ratos , Animais , Humanos , Ligantes , Membrana Celular/metabolismo , Membranas , Aglutininas do Germe de Trigo/metabolismo , Microscopia de Força Atômica/métodos , Proteínas de Membrana/metabolismo
15.
Biotechnol Biofuels Bioprod ; 16(1): 50, 2023 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-36964595

RESUMO

BACKGROUND: 1,3-Propanediol (1,3-PDO) is a platform compound, which has been widely used in food, pharmaceutical and cosmetic industries. Compared with chemical methods, the biological synthesis of 1,3-PDO has shown promising applications owing to its mild conditions and environmental friendliness. However, the biological synthesis of 1,3-PDO still has the problem of low titer and yield due to the shortage of reducing powers. RESULTS: In this study, Klebsiella sp. strain YT7 was successfully isolated, which can synthesize 11.30 g/L of 1,3-PDO from glycerol in flasks. The intracellular redox regulation strategy based on the addition of electron mediators can increase the 1,3-PDO titer to 28.01 g/L. Furthermore, a co-culturing system consisting of strain YT7 and Shewanella oneidensis MR-1 was established, which can eliminate the supplementation of exogenous electron mediators and reduce the by-products accumulation. The 1,3-PDO yield reached 0.44 g/g and the final titer reached 62.90 g/L. The increased titer and yield were attributed to the increased redox levels and the consumption of by-products. CONCLUSIONS: A two-bacterium co-culture system with Klebsiella sp. strain YT7 and S. oneidensis strain MR-1 was established, which realized the substitution of exogenous electron mediators and the reduction of by-product accumulation. Results provided theoretical basis for the high titer of 1,3-PDO production with low by-product concentration.

16.
bioRxiv ; 2023 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-37790307

RESUMO

Multivalency enables nanostructures to bind molecular targets with high affinity. Although antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we design a nanoscale synthetic antibody ("nano-synbody") based on a three-helix bundle DNA nanostructure with one, two, or three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The nano-synbody was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance affinity. The protein-DNA nano-synbody shows tight binding to the wild-type, Delta, and several Omicron variants of the SARS-CoV-2 spike trimer, with affinity increasing as the number of arms increases from one to three. The effectiveness of the nano-synbody was also verified using a pseudovirus neutralization assay, with the three-arm nanostructure inhibiting two Omicron variants against which the structures with only one or two arms are ineffective. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms attached to the RBD domains, confirming the intended trivalent attachment. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach two or more different binding ligands, will enable the high-affinity targeting of a range of proteins not possible with traditional antibodies.

17.
ACS Sens ; 7(9): 2625-2633, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36000947

RESUMO

Separation and identification of different proteins is one of the most fundamental tasks in biochemistry that is typically achieved by electrophoresis and Western blot techniques. Yet, it is challenging to perform such an analysis with a small sample size. Using a principle analogous to these conventional approaches, we present a label-free, single-molecule technique to identify different proteins based on the difference in their size, charge, and antibody binding. We tether single protein molecules to a sensor surface with a flexible polymer and drive them into oscillation by applying an alternating electric field. By tracking the nanometer-scale oscillation of each protein molecule via high-resolution scattering microscopy, the size and charge of each protein molecule can be determined simultaneously. Changes induced by varying the buffer pH and antibody binding are also investigated, which allows us to further expand the separation ability and identify two different proteins in a mixture. We anticipate our technique will contribute to single protein analysis and biosensing.


Assuntos
Microscopia , Proteínas , Nanotecnologia , Polímeros
18.
ACS Sens ; 7(11): 3461-3469, 2022 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-36273329

RESUMO

Most label-free techniques rely on measuring refractive index or mass change on the sensor surface. Thus, it is challenging for them to measure small molecules or enzymatic processes that only induce a minor mass change on the analyte molecules. Here, we have developed a technique by combining Surface Plasmon Resonance sensing with an Oscillating Biomolecule Layer approach (SPR-OBL) to enhance the sensitivity of traditional SPR. In addition to the inherent mass sensitivity, SPR-OBL is also sensitive to the charge and conformational change of the analyte; hence it overcomes the mass limit and is able to detect small molecules. We show that the multimetric SPR-OBL measurement allows for sensing any changes regarding mass, charge, and conformation, which expands the detection capability of SPR.


Assuntos
Refratometria , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Cinética
19.
ACS Cent Sci ; 8(9): 1272-1281, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36188347

RESUMO

Precise and sensitive detection of intracellular proteins and complexes is key to the understanding of signaling pathways and cell functions. Here, we present a label-free single-molecule pulldown (LFSMP) technique for the imaging of released cellular protein and protein complexes with single-molecule sensitivity and low sample consumption down to a few cells per mm2. LFSMP is based on plasmonic scattering imaging and thus can directly image the surface-captured molecules without labels and quantify the binding kinetics. In this paper, we demonstrate the detection principle for LFSMP, study the phosphorylation of protein complexes involved in a signaling pathway, and investigate how kinetic analysis can be used to improve the pulldown specificity. We wish our technique can contribute to uncovering the molecular mechanisms in cells with single-molecule resolution.

20.
Nat Commun ; 13(1): 2298, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35484120

RESUMO

Evanescent illumination has been widely used to detect single biological macromolecules because it can notably enhance light-analyte interaction. However, the current evanescent single-molecule detection system usually requires specially designed microspheres or nanomaterials. Here we show that single protein detection and imaging can be realized on a plain glass surface by imaging the interference between the evanescent lights scattered by the single proteins and by the natural roughness of the cover glass. This allows us to quantify the sizes of single proteins, characterize the protein-antibody interactions at the single-molecule level, and analyze the heterogeneity of single protein binding behaviors. In addition, owing to the exponential distribution of evanescent field intensity, the evanescent imaging system can track the analyte axial movement with high resolution, which can be used to analyze the DNA conformation changes, providing one solution for detecting small molecules, such as microRNA. This work demonstrates a label-free single protein imaging method with ordinary consumables and may pave a road for detecting small biological molecules.


Assuntos
Física , Cinética , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Ligação Proteica
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