Supramolecular Mimotope Peptide Nanofibers Promote Antibody-Ligand Polyvalent and Instantaneous Recognition for Biopharmaceutical Analysis.

Peptide-based supramolecules exhibit great potential in various fields due to their improved target recognition ability and versatile functions. However, they still suffer from numerous challenges for the biopharmaceutical analysis, including poor self-assembly ability, undesirable ligand-antibody binding rates, and formidable target binding barriers caused by ligand crowding. To tackle these issues, a "polyvalent recognition" strategy employing the CD20 mimotope peptide derivative NBD-FFVLR-GS-WPRWLEN (acting on the CDR domains of rituximab) was proposed to develop supramolecular nanofibers for target antibody recognition. These nanofibers exhibited rapid self-assembly within only 1 min and robust stability. Their binding affinity (179 nM) for rituximab surpassed that of the monomeric peptide (7 µM) by over 38-fold, highlighting that high ligand density and potential polyvalent recognition can efficiently overcome the target binding barriers of traditional supramolecules. Moreover, these nanofibers exhibited an amazing "instantaneous capture" rate (within 15 s), a high recovery (93 ± 3%), and good specificity for the target antibody. High-efficiency enrichment of rituximab was achieved from cell culture medium with good recovery and reproducibility. Intriguingly, these peptide nanofibers combined with bottom-up proteomics were successful in tracking the deamidation of asparagine 55 (from 10 to 16%) on the rituximab heavy chain after 21 day incubation in human serum. In summary, this study may open up an avenue for the development of versatile mimotope peptide supramolecules for biorecognition and bioanalysis of biopharmaceuticals.

Co(III)-NTA Mediated Antigen Immobilization on a Fiber Optic-SPR Biosensor for Detection of Autoantibodies in Autoimmune Diseases: Application in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Autoantibodies are key biomarkers in clinical diagnosis of autoimmune diseases routinely detected by enzyme-linked immunosorbent assays (ELISAs). However, the complexity of these assays is limiting their use in routine diagnostics. Fiber optic-surface plasmon resonance (FO-SPR) can overcome these limitations, but improved surface chemistries are still needed to guarantee detection of autoantibodies in complex matrices. In this paper, we describe the development of an FO-SPR immunoassay for the detection of autoantibodies in plasma samples from immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients. Hereto, hexahistidine-tagged recombinant ADAMTS13 (rADAMTS13-His6) was immobilized on nitrilotriacetic acid (NTA)-coated FO probes chelated by cobalt (Co(III)) and exposed to anti-ADAMTS13 autoantibodies. Initial studies were performed to optimize rADAMTS13-His6 immobilization and to confirm the specificity of the immunoassay for detection of anti-ADAMTS13 autoantibodies with FO-SPR. The performance of the immunoassay was then evaluated by comparing Co(III)- and nickel (Ni(II))-NTA stabilized surfaces, confirming the stable immobilization of the antigen in Co(III)-NTA-functionalized FO probes. A calibration curve was prepared with a dilution series of a cloned human anti-ADAMTS13 autoantibody in ADAMTS13-depleted plasma resulting in an average interassay coefficient of variation of 7.1% and a limit of detection of 0.24 ng/mL. Finally, the FO-SPR immunoassay was validated using seven iTTP patient plasma samples, resulting in an excellent correlation with an in-house-developed ELISA (r = 0.973). In summary, the specificity and high sensitivity in combination with a short time-to-result (2.5 h compared to 4-5 h for a regular ELISA) make the FO-SPR immunoassay a powerful assay for routine diagnosis of iTTP and with extension for any other autoimmune disease.

Carbon dots: Principles and their applications in food quality and safety detection.

In the past ten years, as a novel and prospective nanomaterials, carbon dots have acquired tremendous attention for their unique optical and physicochemical properties, high compatibility and low cost, as well as great potential in sensing area. This review aims to present the current detecting principles based on carbon dots and other nano biological technologies, involving fluorescence quenching and recovery mechanisms. The synthetic and modificatory approaches in making carbon dots including top-down and bottom-up methods, as well as surface passivation and heteroatom doping ways are introduced. Their applications in food area, concerning detection of nutrients, restricted or banned substances as well as foodborne pathogenic bacteria and the toxins secreted are discussed. Finally, the difficulties to be overcome or problems to be solved are presented, and other novel techniques to combine with carbon dots to obtain more stable and specific nanosensors in various fields are proposed. Although carbon dots based sensors have shown the potential in sensing aspect of food area, as food samples are complex in compositions that may cause interferences, more novel techniques are needed to combine with carbon dots to develop sensitive and specific sensing probes.

Applications of Computer Vision for Assessing Quality of Agri-food Products: A Review of Recent Research Advances.

With consumer concerns increasing over food quality and safety, the food industry has begun to pay much more attention to the development of rapid and reliable food-evaluation systems over the years. As a result, there is a great need for manufacturers and retailers to operate effective real-time assessments for food quality and safety during food production and processing. Computer vision, comprising a nondestructive assessment approach, has the aptitude to estimate the characteristics of food products with its advantages of fast speed, ease of use, and minimal sample preparation. Specifically, computer vision systems are feasible for classifying food products into specific grades, detecting defects, and estimating properties such as color, shape, size, surface defects, and contamination. Therefore, in order to track the latest research developments of this technology in the agri-food industry, this review aims to present the fundamentals and instrumentation of computer vision systems with details of applications in quality assessment of agri-food products from 2007 to 2013 and also discuss its future trends in combination with spectroscopy.

Applications of near-infrared spectroscopy in food safety evaluation and control: a review of recent research advances.

Food safety is a critical public concern, and has drawn great attention in society. Consequently, developments of rapid, robust, and accurate methods and techniques for food safety evaluation and control are required. As a nondestructive and convenient tool, near-infrared spectroscopy (NIRS) has been widely shown to be a promising technique for food safety inspection and control due to its huge advantages of speed, noninvasive measurement, ease of use, and minimal sample preparation requirement. This review presents the fundamentals of NIRS and focuses on recent advances in its applications, during the last 10 years of food safety control, in meat, fish and fishery products, edible oils, milk and dairy products, grains and grain products, fruits and vegetables, and others. Based upon these applications, it can be demonstrated that NIRS, combined with chemometric methods, is a powerful tool for food safety surveillance and for the elimination of the occurrence of food safety problems. Some disadvantages that need to be solved or investigated with regard to the further development of NIRS are also discussed.

Biomimetic affinity membrane roll column for rapid purification of C-reactive protein.

To in-depth explore the action mechanism of C-reactive protein (CRP) and precisely study its signaling pathways, it is essential to acquire high-purity CRP while preserving its intact structure and functionality. In this study, we propose and fabricate a high-density 2-methacryloyloxyethyl phosphorylcholine (MPC)-modified membrane roll column (MPC-MRC) using a surface-initiated atom transfer radical polymerization (SI-ATRP) approach, which can overcome these limitations (long incubation time and low adsorption capacity) of conventional enrichment materials. The MPC-MRC incorporates a high-density 2-hydroxyethyl methacrylate polymer brush to prevent non-specific protein adsorption and multiple MPC polymer brush layers for high-performance enrichment of CRP in the company of calcium ions. Furthermore, the MPC-MRC exhibits high permeability, hydrophilicity, and mechanical strength. Compared to previous technologies, this novel material demonstrates significantly higher CRP binding capacity (310.3 mg/g), shorter processing time (only 15 min), and lower cost (only 12 USD/column). Notably, the MPC-MRC enables fast and effective purification of CRP from both human and rat serum, exhibiting good selectivity, recovery (> 91.3 %), and purity (> 95.2 %). Thus, this proposed purification approach based on MPC-MRC holds great potential for target protein enrichment from complex samples, as well as facilitating in-depth studies of its biological functions.

Tetrapeptide-based mimotope affinity monolith for the enrichment and analysis of anti-HER2 antibody and antibody-drug conjugate.

Selective enrichment and analysis of therapeutic antibodies in biological fluids are crucial for the development of biopharmaceuticals. Recently, peptide-based affinity chromatography has exhibited fascinating prospects for antibody enrichment due to the high affinity and specificity of small peptides. However, the post-modification approach of peptide ligands on the material surface is complicated and time-consuming. In this study, a methacrylate modified tetrapeptide (m-EDPW) was firstly demonstrated as the affinity ligand of trastuzumab (Kd = 1.91 ± 1.81 µM). Next, the m-EDPW based affinity monolith was prepared using a facile one-step polymerization method, which could overcome the drawbacks of traditional post-modification preparation strategies. Based on the monolith as described above, a simple enrichment approach was developed under the optimal washing and elution conditions. Based on the excellent properties, such as high porosity (53.09%), weak electrostatic interaction and suitable affinity (1.00 ± 2.14 µM for anti-HER2 ADC), this novel monolith exhibited good specificity and recovery for antibodies (91.6% for trastuzumab, 98.37% for anti-HER2 ADC), and low nonspecific adsorption for human serum albumin (DBC10% = 0.5 mg/g polymer). Particularly, this material was successfully applied to enrich trastuzumab and its related antibody-drug conjugate (ADC) from different cell culture medias. The dynamic tracking analysis of ADC in the critical quality attributes (e.g., charge variants, drug to antibody ratio and subunit conjugation ratio) was also achieved by combining the enrichment approach, capillary electrophoresis or reversed phase liquid chromatography. In summary, the exploited peptide-based mimotope affinity materials showed a great potential for the application in biopharmaceutical analysis.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood.

The ongoing COVID-19 pandemic has emphasized the urgent need for rapid, accurate, and large-scale diagnostic tools. Next to this, the significance of serological tests (i.e., detection of SARS-CoV-2 antibodies) also became apparent for studying patients' immune status and past viral infection. In this work, we present a novel approach for not only measuring antibody levels but also profiling of binding kinetics of the complete polyclonal antibody response against the receptor binding domain (RBD) of SARS-CoV-2 spike protein, an aspect not possible to achieve with traditional serological tests. This fiber optic surface plasmon resonance (FO-SPR)-based label-free method was successfully accomplished in COVID-19 patient serum and, for the first time, directly in undiluted whole blood, omitting the need for any sample preparation. Notably, this bioassay (1) was on par with FO-SPR sandwich bioassays (traditionally regarded as more sensitive) in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) had a significantly shorter time-to-result of only 30 min compared to >1 or 4 h for the FO-SPR sandwich bioassay and the conventional ELISA, respectively. Finally, the label-free approach revealed that no direct correlation was present between antibody levels and their kinetic profiling in different COVID-19 patients, as another evidence to support previous hypothesis that antibody-binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Taking all this into account, the presented work positions the FO-SPR technology at the forefront of other COVID-19 serological tests, with a huge potential toward other applications in need for quantification and kinetic profiling of antibodies.

Point-of-care therapeutic drug monitoring of adalimumab by integrating a FO-SPR biosensor in a self-powered microfluidic cartridge.

Disease treatment with advanced biological therapies such as adalimumab (ADM), although largely beneficial, is still costly and suffers from loss of response. To tackle these aspects, therapeutic drug monitoring (TDM) is proposed to improve treatment dosing and efficacy, but is often associated with long sampling-to-result workflows. Here, we present an in-house constructed ADM-sensor, allowing TDM of ADM at the doctor's office. This biosensor brings fiber optic surface plasmon resonance (FO-SPR), combined with self-powered microfluidics, to a point of care (POC) setting for the first time. After developing a rapid FO-SPR sandwich bioassay for ADM detection on a commercial FO-SPR device, this bioassay was implemented on the fully-integrated ADM-sensor. For the latter, we combined (I) a gold coated fiber optic (FO) probe for bioassay implementation and (II) an FO-SPR readout system with (III) the self-powered iSIMPLE microfluidic technology empowering plasma sample and reagent mixing on the-cartridge as well as connection to the FO-SPR readout system. With a calculated limit of detection (LOD) of 0.35 µg/mL in undiluted plasma, and a total time-to-result (TTR) within 12 min, this innovative biosensor demonstrated a comparable performance to existing POC biosensors for ADM quantification in patient plasma samples, while requiring only 1 µL of plasma. Whereas this study demonstrates great potential for FO-SPR biosensing at the POC using ADM as a model case, it also shows huge potential for bedside TDM of other drugs (e.g. other immunosuppressants, anti-epileptics and antibiotics), as the bioassay is highly amenable to adaptation.

Gold nanoparticle enhanced multiplexed biosensing on a fiber optic surface plasmon resonance probe.

We present an innovative multiplexing concept on a fiber optic surface plasmon resonance (FO-SPR) platform and demonstrate for the first time the simultaneous detection of two targets using the same FO sensor probe. Co(III)-NTA chemistry was used for oriented and stable co-immobilization of two different His6-tagged bioreceptors. T2C2 and MDTCS (i.e. fragments of the ADAMTS13 metalloprotease linked to the thrombotic thrombocytopenic purpura disorder) served as model system bioreceptors together with their respective targets (4B9 and II-1 antibodies). Gold nanoparticles were used here in an original way for discriminating the two targets in the same sample, in addition to their traditional signal amplification-role. After verifying the specificity of the selected model system, we studied the bioreceptor surface density and immobilization order. Innovative approach to lower the bioreceptor concentration below surface saturation resulted in an optimal detection of both targets, whereas the order of immobilization of the two bioreceptors did not give any significant difference. By sequentially immobilizing the T2C2 and MDTC bioreceptors, we established calibration curves in buffer and 100-fold diluted human blood plasma. This resulted in calculated limits of detection of 3.38 and 2.31 ng/mL in diluted plasma for 4B9 and II-1, respectively, indicating almost the same sensitivity as in buffer. Importantly, we also proved the applicability of the established calibration curves for quantifying the targets at random and more realistic ratios, directed by the design of experiments. This multiplexing study further expands the repertoire of applications on the FO-SPR biosensing platform, which together with its intrinsic features opens up great opportunities for diagnostics and life sciences.

Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry.

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

Advancements in SPR biosensing technology: An overview of recent trends in smart layers design, multiplexing concepts, continuous monitoring and in vivo sensing.

Inspired by the rapid progress and existing limitations in surface plasmon resonance (SPR) biosensing technology, we have summarized the recent trends in the fields of both chip-SPR and fiber optic (FO)-SPR biosensors during the past five years, primarily regarding smart layers design, multiplexing, continuous monitoring and in vivo sensing. Versatile surface chemistries, biomaterials and nanomaterials have been utilized thus far to generate smart layers on SPR platforms and as such achieve oriented immobilization of bioreceptors, improved fouling resistance and sensitivity enhancement, collectively aiming to improve the biosensing performance. Furthermore, often driven by the desires for time- and cost-effective quantification of multiple targets in a single measurement, efforts have been made to implement multiplex bioassays on SPR platforms. While this aspect largely remains difficult to attain, numerous alternative strategies arose for obtaining parallel analysis of multiple analytes in one single device. Additionally, one of the upcoming challenges in this field will be to succeed in using SPR platforms for continuous measurements and in vivo sensing, and as such match up other biosensing platforms where these goals have been already conquered. Overall, this review will give insight into multiple possibilities that have become available over the years for boosting the performance of SPR biosensors. However, because combining them all into one optimal sensor is practically not feasible, the final application needs to be considered while designing an SPR biosensor, as this will determine the requirements of the bioassay and will thus help in selecting the essential elements from the recent progress made in SPR sensing.

Expanding a Portfolio of (FO-) SPR Surface Chemistries with the Co(III)-NTA Oriented Immobilization of His6-Tagged Bioreceptors for Applications in Complex Matrices.

Cobalt-nitrilotriacetic acid (Co(III)-NTA) chemistry is a recognized approach for oriented patterning of His6-tagged bioreceptors. We have applied the matching strategy for the first time on a surface plasmon resonance (SPR) platform, namely, the commercialized fiber optic (FO)-SPR. To accomplish this, His6-tagged bioreceptor (scFv-33H1F7) and its target PAI-1 were used as a model system, after scrutinizing the specificity of their interaction. When benchmarked to traditional carboxyl-based self-assembled monolayers (SAM), NTA allowed (1) more efficient FO-SPR surface coverage with bioreceptors compared with the former and (2) realization of thus far difficult-to-attain label-free bioassays on the FO-SPR platform in both buffer and 20-fold diluted human plasma. Moreover, Co(III)-NTA surface proved to be compatible with traditional gold nanoparticle-mediated signal amplification in the buffer as well as in 10-fold diluted human plasma, thus expanding the dynamic detection range to low ng/mL. Both types of bioassays revealed that scFv-33H1F7 immobilized on the FO-SPR surface using different concentrations (20, 10, or 5 µg/mL) had no impact on the bioassay sensitivity, accuracy, or reproducibility despite the lowest concentration effectively resulting in close to 20% fewer bioreceptors. Collectively, these results highlight the importance of Co(III)-NTA promoting the oriented patterning of bioreceptors on the FO-SPR sensor surface for securing robust and sensitive bioassays in complex matrices, both in label-free and labeled formats.