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The Antibody-Free Recognition of Cancer Cells Using Plasmonic Biosensor Platforms with the Anisotropic Resonant Metasurfaces
摘要： It is vital and promising for portable and disposable biosensing devices to achieve on-site detection and analysis of cancer cells. Although traditional labelling techniques provide an accurate quantitative measurement, the complicated cell staining and high-cost measurements limit its further development. Here, we demonstrate a non-immune biosensing technology. The plasmonic biosensors which is based on anisotropic resonant split ring resonators in terahertz range successfully realize the antibody-free recognition of cancer cells. The dependences of Δf and fitted phase slope (FPS) on the cancer cell concentration at different polarizations give new perspective in hexagonal radar maps. The results indicate that the lung cancer cell A549 and liver cancer cell HepG2 can be distinguished and determined simply based on the enclosed shapes in the radar maps without any antibody introduction. The minimum concentration of identification reduces as low as 1×104 cells/ml and such identification can be kept valid in a large range of cell concentration, ranging from 104 to 105. The construction of two-dimensional extinction intensity cards of corresponding cancer cells based on the wavelet transform method also supplies corresponding information for the antibody-free recognition and determination of two cancer cells. Our plasmonic MBs show a great potential in the determination and recognition of label-free cancer cells, being an alternative to non-immune biosensing technology.
关键词： terahertz,antibody-free biosensing,cancer cells,metasurfaces,continuous wavelet transform
更新于2025-09-23 15:19:57
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Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots
摘要： Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10? 7 and 1.35 × 10? 8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immuno-magnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.
关键词： single-domain antibody,immunomagnetic nanobeads,CSFV,classical swine fever,quantum dots
更新于2025-09-23 15:19:57
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Impact of Tryptophan Oxidation in Complementarity-Determining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance
摘要： Purpose Tryptophan’s (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. Methods Selective Trp oxidation was induced by 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. Results Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. Conclusions Trp oxidation in CDRs can adversely impact mAbs’ conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.
关键词： Tryptophan oxidation,surface plasmon resonance,complementarity-determining region,hydrogen-deuterium exchange mass spectrometry,monoclonal antibody
更新于2025-09-23 15:19:57
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Development, optimization and structural characterization of an efficient peptide-based photoaffinity crosslinking reaction for generation of homogeneous conjugates from wild-type antibodies
摘要： Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein, we report the development and detailed characterization of a robust photoaffinity crosslinking method for site-specific conjugation to fully-glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously-described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. First, we describe the initial discovery of an effective Bpa mutant peptide and optimization of reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody. Second, we assessed the scope of the photoconjugation reaction across different human and non-human antibodies and antibody mutants. Third, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and show that the resulting conjugate is both stable in plasma and as potent as a conventional antibody drug conjugate in cells, portending well for future biological applications.
关键词： wild-type antibodies,Fc domain,photoaffinity crosslinking,site-specific conjugation,antibody-drug conjugates
更新于2025-09-23 15:19:57
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Determination of the Concentration of Polyamines with SPR-based Immune Biosensor for Early Diagnostics of Breast Cancer
摘要： The paper presents the results of research on the development of immune biosensor test system for express detection of polyamines in cells of breast cancer. Determination of polyamines was performed by using an analytical device - immune biosensor based on surface plasmon resonance (SPR), where "antigen-antibody" reaction is performed in real time on the surface of transducer, resulting in formation of immune complexes and recording the shift of resonance angle. The method can detect the studied polyamines spermine and spermidine in concentration less than 10 ng in 1 mL, and with increasing concentrations a statistical probability of the analysis' result rise sharply. Moreover, the dependence of sensitivity of biosensor response from the concentration of polyamines is in the range of 10-100 ng /mL.
关键词： Antigen,Antibody,Breast cancer,Surface plasmon resonance,Polyamines
更新于2025-09-19 17:15:36
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P-glycoprotein targeted photodynamic therapy of chemoresistant tumors using recombinant Fab fragment conjugates
摘要： P-glycoprotein (Pgp) has been considered as a major cause of cancer multidrug resistance; however, clinical solutions to overcome this drug resistance do not exist despite the tremendous endeavors. The lack of cancer specificity is a main reason for clinical failure of conventional approaches. Targeted photodynamic therapy (PDT) is highly cancer specific by combining antibody targeting and locoregional light irradiation. We aimed to develop Pgp-targeted PDT using antibody–photosensitizer conjugates made of a recombinant Fab fragment. We prepared the photosensitizer conjugates by expressing a recombinant Fab fragment and specifically linking IR700-maleimide at the C-terminal of the Fab heavy chain. In vitro studies showed that the Fab conjugates specifically bind to Pgp. Their phototoxicity was comparable to full antibody conjugates when assayed with conventional 2-D cell culture, but they outperformed the full antibody conjugates in a 3-D tumor spheroid model. In a mouse xenograft model of chemoresistant tumors, Fab conjugates showed Pgp specific delivery to chemoresistant tumors. Upon irradiation with near-infrared light, they caused rapid tumor shrinkage and significantly prolonged the survival of tumor-bearing mice. Compared to the full antibody conjugates, Fab conjugates took a shorter time to reach peak tumor levels and achieved a more homogeneous tumor distribution. This allows light irradiation to be initiated at a shorter time interval after the conjugate injection, and thus may facilitate clinical translation. We conclude that our targeted PDT approach provides a highly cancer-specific approach to combat chemoresistant tumors, and that the conjugates made of recombinant antibody fragments are superior to full antibody conjugates for targeted PDT.
关键词： chemoresistance,P-glycoprotein,recombinant Fab fragment,photodynamic therapy,antibody conjugates
更新于2025-09-19 17:15:36
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Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages
摘要： Purpose: Macrophage mannose receptor (MMR, CD206) expressing tumor-associated macrophages (TAM) are protumorigenic and was reported to negatively impact therapy responsiveness and is associated with higher chances of tumor relapse following multiple treatment regimens in preclinical tumor models. Since the distribution of immune cells within the tumor is often heterogeneous, sampling errors using tissue biopsies will occur. In order to overcome this limitation, we propose positron emission tomography (PET)/X-ray computed tomography (CT) imaging using 68Ga-labeled anti-MMR single-domain antibody fragment (sdAb) to assess the presence of these protumorigenic TAM. Procedures: Cross-reactive anti-MMR-sdAb was produced according to good manufacturing practice (GMP) and conjugated to p-SCN-Bn-NOTA bifunctional chelator for 68Ga-labeling. Biodistribution and PET/CT studies were performed in wild-type and MMR-deficient 3LL-R tumor-bearing mice. Biodistribution data obtained in mice were extrapolated to calculate radiation dose estimates for the human adult using OLINDA software. A 7-day repeated dose toxicity study for NOTA-anti-MMR-sdAb was performed in healthy mice up to a dose of 1.68 mg/kg. Results: [68Ga]Ga-NOTA-anti-MMR-sdAb was obtained with 76 ± 2 % radiochemical yield, 99 ± 1 % radiochemical purity, and apparent molar activity of 57 ± 11 GBq/μmol. In vivo biodistribution analysis showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor, with tumor-to-blood and tumor-to-muscle ratios of 6.80 ± 0.62 and 5.47 ± 1.82, respectively. The calculated effective dose was 0.027 mSv/MBq and 0.034 mSv/MBq for male and female, respectively, which means that a proposed dose of 185 MBq in humans would yield a radiation dose of 5.0 and 6.3 mSv to male and female patients, respectively. In the toxicity study, no adverse effects were observed. Conclusions: Preclinical validation of [68Ga]Ga-NOTA-anti-MMR-sdAb showed high specific uptake of this tracer in MMR-expressing TAM and organs, with no observed toxicity. [68Ga]Ga-NOTA-anti-MMR-sdAb is ready for a phase I clinical trial.
关键词： Single-domain antibody (sdAb),PET,Tumor-associated macrophages (TAM),Macrophage mannose receptor (MMR)
更新于2025-09-19 17:15:36
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Lab-on-tip: Protruding-shaped all-fiber plasmonic microtip probe toward in-situ chem-bio detection
摘要： Lab-on-tip technology opens a new door for optical fiber surface plasmon resonance (SPR) sensors. In this paper, we first demonstrate an ultra-compact reflective SPR microtip probe on the flat fiber end in experiment, which has a length of tens of microns and a diameter of several microns. Simulations and experiments show that this new fiber SPR microtip probe coated with a nano-goldfilm has excellent refractive index (RI) sensing characteristic and great potential for biochemical detection at single cell level. Based on the high RI sensitivity, a bio-probe assembled with Chitosan/Polysodium Styrene Sulfonate (Cs/PSS) nano-polyelectrolyte film and the sheep anti-human IgG-HRP is prepared to recognize human IgG specifically. Due to the high surface sensitivity, the SPR microtip probe exhibits an ultra-low detection limit for human IgG. Due to the high bulk sensitivity and high thermo-optical coefficient (TOC) of polydimethylsiloxane (PDMS), a reference temperature probe based on SPR microtip is proposed, which is used to compensate for the temperature cross-sensitivity of the bio-probe. This new SPR microtip probe can be used not only in optical fiber sensor, but also in Tip-Enhanced Near-Field Raman Microscopy and Scanning Near-Field Optical Microscope.
关键词： Surface plasmon resonance,Optical fiber microtip,Refractive index detection,Antibody detection
更新于2025-09-19 17:13:59
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[IEEE 2019 IEEE International Conference on Computation, Communication and Engineering (ICCCE) - Fujian P.R, China (2019.11.8-2019.11.10)] 2019 IEEE International Conference on Computation, Communication and Engineering (ICCCE) - The IR Drop Effect of 2T1C and 3T1C Driving Modes in Top Emission AMOLED
摘要： The immune system in homo sapiens protects the body against diseases by identifying and attacking foreign pathogens. However, when the system misidenti?es native cells as threats, it results in an auto-immune response. The auto-antibodies generated during this phenomenon may be identi?ed through the indirect immuno?uorescence test. An important constituent process of this test is the automated identi?cation of antigen patterns in the cell images, which is the focus of this research. We perform a detailed literature review and present a framework to automate the identi?cation of antigen patterns. The ef?cacy of the framework, demonstrated on the MIVIA ICPR 2012 HEp-2 Cell Contest and SNP HEp-2 Cell datasets, suggests that the algorithm is comparable with the state-of-the-art approaches.
关键词： anti-nuclear antibody testing,Biomedical imaging,HEp-2 cells,laws texture measure,indirect immuno?uorescence test
更新于2025-09-19 17:13:59
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A fluorescent plasmonic biochip assay for multiplex screening of diagnostic serum antibody targets in human Lyme disease
摘要： Lyme disease (LD) diagnosis using the current two-tier algorithm is constrained by low sensitivity for early-stage infection and ambiguity in determining treatment response. We recently developed a protein microarray biochip that measures diagnostic serum antibody targets using grating-coupled fluorescent plasmonics (GC-FP) technology. This strategy requires microliters of blood serum to enable multiplexed biomarker screening on a compact surface and generates quantitative results that can be further processed for diagnostic scoring. The GC-FP biochip was used to detect serum antibodies in patients with active and convalescent LD, as well as various negative controls. We hypothesized that the quantitative, high-sensitivity attributes of the GC-FP approach permit: 1) screening of antibody targets predictive for LD status, and 2) development a diagnostic algorithm that is more sensitive, specific, and informative than the standard ELISA and Western blot assays. Notably, our findings led to a diagnostic algorithm that may be more sensitive than the current standard for detecting early LD, while maintaining 100% specificity. We further show that analysis of relative antibody levels to predict disease status, such as in acute and convalescent stages of infection, is possible with a highly sensitive and quantitative platform like GC-FP. The results from this study add to the urgent conversation regarding better diagnostic strategies and more effective treatment for patients affected by tick-borne disease.
关键词： Lyme disease,grating-coupled fluorescent plasmonics (GC-FP) technology,diagnostic serum antibody targets,multiplex screening,fluorescent plasmonic biochip assay
更新于2025-09-19 17:13:59