Anti-fouling of material surfaces is a major concern in the performance of biomedical materials in biological environments. Surface modification of polymeric materials is an effective strategy for controlling the anti-fouling properties of biomedical materials. Synthetic polymers such as poly(ethylene glycol) (PEG), which exhibit high bioinertness and anti-fouling properties, are widely used. Peptide-based anti-fouling materials are gaining attention because of their biocompatibility, long-term biodegradability, and structural diversity. In this study, we explored the potential of antifreeze protein type I (AFP) from winter flounder as a peptide-based anti-fouling material. AFP possesses a simple α-helical structure of approximately 30 amino acid residues and presents an amphiphilic interface consisting of an ice-binding surface and a hydrophilic surface. We constructed a recombinant AFP that can be modified on a glass substrate and evaluated its anti-fouling properties. The AFP-modified substrates significantly suppressed the adsorption of plasma proteins, such as human albumin, fibrinogen, and fibronectin. Furthermore, adhesion of human ovarian cancer SKOV3 cells and mouse platelets to the substrates was suppressed by AFP modification. Notably, tandem-repeat AFP constructs exhibited high resistance to proteolytic enzymes, such as trypsin. These findings highlight the potential of recombinant AFP as a potential peptide-based anti-fouling material for biomedical applications.

Thermoresponsive elastin-like polypeptide (ELP) assemblies are promising platforms for functional nanoparticles, but the elevated temperatures required for self-assembly can inactivate heat-labile proteins of interest (POIs). To address this issue, we introduced a genetically encoded SnoopTag/SnoopCatcher cyclization module into an ELP-poly(aspartic acid) (ELP-D) scaffold. Using Renilla luciferase (RLuc) as a model POI, tail-to-side-chain cyclization increased both melting temperature and half-inactivation temperature by ∼6°C, indicating enhanced thermal stability. This stabilization was preserved upon fusion to ELP-D without affecting nanoparticle size or morphology. ELP-D-cRLuc nanoparticles retained ∼70% activity after heating to 42°C, compared with ∼30% for non-cyclized constructs. These results demonstrate that genetically encoded cyclization effectively protects POIs during heat-triggered nanoparticle assembly and enables activity retention at elevated temperatures.

Lipid carriers, such as liposomes and exosomes, are nanoscale vesicular structures composed of phospholipids, glycolipids, membrane proteins, and cholesterol, which have been widely investigated as drug- and nucleic-acid-delivery systems. Functionalization of lipid carrier surfaces with ligand molecules is an effective approach for the targeted delivery of lipid carriers to specific tissues and cells. In this regard, the use of DNA modification represents a rational and promising approach as DNA aptamers exhibit high affinity and specificity for target molecules and offer several advantages over conventional ligands. The current strategies for modifying lipid carriers with DNA remain suboptimal, leaving room for further improvement. In this study, we aimed to modify lipid carriers with single-stranded (ss)DNA-conjugated fusion proteins that have cholesterol-binding abilities. The fusion protein (Rep-ALOD4) was composed of a replication initiator protein derived from porcine circovirus type 2 (Rep), which acted as an ssDNA-conjugating domain, and domain 4 of anthrolysin O (ALOD4), which acted as a cholesterol-binding domain. As Rep-ALOD4 can bind to cholesterol in lipid membranes under mild conditions, ssDNA-conjugated Rep-ALOD4 achieved ssDNA modification on liposome carriers containing more than 30 wt % cholesterol. Furthermore, cell-selective targeting using DNA aptamer- and ssDNA-modified liposomes and exosomes was successful because of the functional applicability of ssDNA-displaying Rep-ALOD4. Thus, the DNA modification platform of Rep-ALOD4 has the potential to substantially advance targeted drug delivery using lipid carriers.

Temperature-responsive elastin-like polypeptides (ELPs) exhibit a low critical solution temperature-type phase transition and offer potential as useful materials for the construction of nanoparticles. Herein, we developed a novel decoration method for ELP-based nanoparticles via isopeptide bond formation with the SnoopTag/SnoopCatcher system that is not affected by the heating process required for particle formation. A mixture of a fusion protein of ELP and poly(aspartic acid) (poly(D)), known as ELP-poly(D), and ELP-poly(D) fused with SnoopCatcher (ELP-poly(D)-SnC) formed protein nanoparticles as a result of the temperature responsiveness of ELP, with the resultant nanoparticles displaying the SnoopCatcher binding domain on their surfaces. In the present study, two model proteins fused to SnoopTag were displayed on the surfaces of protein nanoparticles constructed from ELP-poly(D)-SnC and ELP-poly(D). The model proteins are enhanced green fluorescent protein (EGFP) and Renilla luciferace (Rluc), which exhibits luminescent capability and weak thermostability, respectively. EGFP on the particle surface was found to retain 48.7% activity, while Rluc exhibited almost full activity, as calculated from the binding efficiency and nanoparticle activities recovered after purification. ELP-based nanoparticles containing the SnoopTag/SnoopCatcher system offer the opportunity for particle decoration with a wide range of functional proteins via isopeptide bond formation.

Injectable hydrogels are promising biomaterials for tissue engineering applications due to their ability to deliver bioactive compounds or cells with minimal invasiveness. Temperature-responsive in situ gelling hydrogels, which undergo transition from liquid to gel in response to temperature stimuli, are desirable candidates for injectable hydrogels. Elastin-like polypeptides (ELPs) are well-known temperature-responsive biomaterials for cell scaffolds, drug delivery, and tissue engineering, due to their biocompatibility, biodegradability, and tunable mechanical properties. However, due to high hydrophobicity and heterogeneous aggregation, the development of injectable hydrogel-derived ELPs remains limited. In our previous study, we designed coiled-coil unit-bound ELPs (CUBEs) hydrogel systems, which integrate ELPs, a polyaspartic acid (polyD) chain, a functional peptide, and a coiled-coil peptide. In this study, we evaluated the injectability and cell delivery potential of a basic CUBE hydrogel system, called O-CUBE (AVGVP)42-D88-CL. The O-CUBE protein solution was mixed with human cervical cancer (HeLa) cells, serving as a cell model, and subsequently injected into culture medium pre-warmed to 37 °C to initiate in situ gelation. O-CUBE protein was successfully gelled at an approximately 90 % gelation rate after injection at 37 °C within pH ranges of 6-8. Encapsulated HeLa cells exhibited spheroid morphology, indicating that the hydrogel facilitated cell-cell interactions in three-dimensional culture. Further evaluation using a DNA assay revealed that HeLa cells can survive and proliferate within the hydrogel. These results demonstrate that the CUBE hydrogel system is a promising candidate to deliver cells with minimal invasiveness.

Isolation of specific cells from biological samples is an important aspect of various biological research and diagnostic applications. Magnetic separation using magnetic particles (MPs) allows for easy and specific isolation of the target cells. However, depending on the target cell antigen, biological ligands, such as antibodies, must be modified or altered on MPs. Additionally, further biological evaluation of isolated cells requires the removal of MPs from cells by the enzymatic degradation of the biological ligands. In this study, we designed a magnetic cell separation system in which temperature-responsive protein nanoparticles mediated the interaction between target cells and MPs, achieving the easy changeability of biological ligands, removal of MPs by cooling, and effective cell isolation. The protein nanoparticles were thermally responsively formed from fusion proteins constituted of elastin-like polypeptide (ELP), poly(aspartic acid) [poly(d)], and proteins-of-interest such as NanoLuc luciferase (Nluc) fused with replication initiation protein (Rep) (ELP-poly(d)-Nluc-Rep) or biotin acceptor peptide (BAP) (ELP-poly(d)-Nluc-BAP). Rep exhibited enzymatic conjugation activity with an optional DNA aptamer to protein nanoparticles. The transmembrane glycoprotein mucin 1 (MUC1)-binding DNA aptamer was conjugated to Rep as a model aptamer. Bioluminescence signals emitted from the Nluc domains were used to analyze the binding abilities. BAP contributed to binding to streptavidin-modified MPs via a biotin-streptavidin interaction. The MUC1-conjugated protein nanoparticles bound to MUC1-positive human breast cancer MCF-7 cells via MUC1 aptamers and streptavidin-conjugated MPs via BAP, leading to magnetic cell separation. The ratio of isolated MCF-7 cells via magnetic separation was 71.3% for the MCF-7 suspension at 1000 cells/1 mL. The MPs bound on recovered MCF-7 cells were removed by cooling at 4 °C to induce the dissociation of protein nanoparticles. Magnetic cell separation systems that use protein nanoparticles are a promising technology for biological research and diagnostic applications.

The development of bone implants through bioinspired immobilization of growth factors remains a key issue in the generation of biological interfaces, especially in enhancing osteodifferentiation ability. In this study, we developed a strategy for surface functionalization of poly(lactide-glycolide) (PLGA) and hydroxyapatite (HA) composite substrates through site-specific conjugation of bone morphogenetic protein 2 containing 3,4-hydroxyphenalyalanine (DOPA-BMP2) mediated by tyrosinase and sortase A (SrtA). Firstly, the growth factor BMP2-LPETG containing LPETG motif was successfully expressed in Escherichia coli through recombinant DNA technology. The excellent binding affinity of binding growth factor (DOPA-BMP2) was achieved by converting the tyrosine residue (Y) of YKYKY-GGG peptide into DOPA (X) by tyrosinase, which bound to the substrates. Then its GGG motif was specifically bound to the end of BMP2-LPETG mediated by SrtA. Therefore, the generated bioactive DOPA-BMP2/PLGA/HA substrates significantly promoted the osteogenic differentiation of MC3T3-E1 cells. Thanks to this microbial-assisted engineering approach, our work presents a facile and highly site-specific strategy to engineer biomimetic materials for orthopedics and dentistry by effectively delivering growth factors, peptides, and other biomacromolecules.

DNA modification of the plasma membrane is an excellent approach for controlling membrane-protein interactions, modulating cell-cell/cell-biomolecule interactions, and extending the biosensing field. The hydrophobic insertion of DNA conjugated with hydrophobic anchoring molecules is utilized for tethering DNA on the cell membrane. In this study, we developed an alternative approach to tether DNA on the plasma membrane based on ssDNA- and cholesterol-binding proteins. We designed a fusion protein (Rep-ALOD4) composed of domain 4 of anthrolysin O (ALOD4), which binds to cholesterol in the plasma membrane, and a replication initiator protein derived from porcine circovirus type 2 (Rep), which forms covalent bonds with single-stranded DNA (ssDNA) with a Rep recognition sequence. Rep-ALOD4 conjugates ssDNA to Rep and binds to the plasma membrane via cholesterol, thus tethering ssDNA to the cells. Quartz crystal microbalance measurements showed that membrane cholesterol binding of Rep-ALOD4 to the lipid bilayer containing cholesterol was accelerated above 20% (w/w) cholesterol in the lipid bilayer. Rep-ALOD4 was conjugated to fluorescein-labeled ssDNA (S-FITC-Rep-ALOD4) and used to treat human cervical tumor HeLa cells. The green signal assigned to S-FITC-Rep-ALOD4 was detected along HeLa cells, whereas diminished by cholesterol removal with methyl β-cyclodextrins. Moreover, ssDNA-conjugated Rep-ALOD4 tethered ssDNA-conjugated functional proteins on the HeLa cell plasma membrane via complementary base pairing. Collectively, Rep-ALOD4 has the potential as an ssDNA-tethering material via plasma membrane cholesterol to extend cell surface engineering.

Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(D)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(D)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(D)-Rep and ELP-poly(D)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

Hypothesis: Liposomes coated with long polysarcosine (PSar) chains at a high density might enable long blood circulation and attenuate accelerated blood clearance (ABC) phenomenon.Experiments: In this study, we controlled the length (23, 45, 68 mers) and density (5, 10, 15 mol%) of PSar on liposomal coatings and, furthermore, investigated the effects of PSar length and density on the blood circulation time, biodistribution, immune response, and ABC phenomenon induction. Length-controlled PSar-bound lipids (PSar-PEs) were synthesized using a click reaction and inserted into bare liposomes at different combinations of chain lengths and proportions.Findings: Although all PSar-coated liposomes (PSar-lipos) had similar morphological, physical, and chemical properties, they had different blood circulation times and biodistribution, and exerted varied effects on the immune system. All PSar-lipos with different PSar length and density showed a similar anti-PSar IgM response. Liposomes modified with the longest PSar chain (68 mers) at a high density (15 mol%) showed the longest blood circulation time and, additionally, attenuated ABC phenomenon compared with PEG-lipo. The ex vivo analysis of the biodistribution of liposomes revealed that a thick PSar layer enhanced the blood circulation time of liposomes due to the reduction of the accumulation of liposomes in the liver and spleen. These findings provide new in-sights into the relationship between IgM expression and ABC phenomenon inhibition.

Background Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions. Objective We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system. Method Naso-oropharyngeal samples of 339 COVID-19 suspected cases were collected and evaluated through RT-qPCR that were stored up to 30 days in different conditions (i.e. -80 degrees C, -20 degrees C and initially at 4 degrees C followed by -80 degrees C). The clinical specimens were evaluated with our in-house ELISA system after finalizing the assay method through checkerboard assay and minimizing the signal/noise ratio. Result The ELISA system showed the highest sensitivity (92.9%) for samples with Ct <= 30 and preserving at -80 degrees C temperature. The sensitivity reduced proportionally with increasing Ct value and preserving temperature. However, the specificity ranged between 98.3% and 100%. Conclusion The results indicate the necessity of early infection phase diagnosis and lower temperature preservation of samples to perform rapid antigen ELISA tests.

Protein nanoparticles are potentially valuable carriers for drug delivery, because they can be easily designed to perform various functions such as targeting to specific cells and penetration of cells and tissues.

BACKGROUND:Vaccination with the Oxford-AstraZeneca COVID-19 vaccine (AZD1222) initially started in the UK and quickly implemented around the Globe, including Bangladesh. Up to date, more than nine million doses administrated to the Bangladeshi public.METHOD:Herein, we studied the antibody response to the first dose of AZD1222 in 86 Bangladeshi individuals using in-house ELISA kits. Study subjects were categorized into two groups, convalescent and uninfected, based on prior infection history and SARS-CoV-2 nucleocapsid-IgG profiles.RESULTS:All the convalescent individuals presented elevated spike-1-IgG compared to 90% of uninfected ones after the first dose. Day >28 post-vaccination, the convalescent group showed six times higher antibody titer than the uninfected ones. The most elevated antibody titers for the former and later group were found at Day 14 and Days >28 post-vaccination, respectively. The spike-1-IgA titer showed a similar pattern as spike-1-IgG, although in a low-titer. In contrast, the IgM titer did not show any significant change in either group.CONCLUSION:High antibody titer in the convalescent group, signify the importance of the first dose among the uninfected group. This study advocates the integration of antibody tests in vaccination programs in the healthcare system for maximizing benefit.

Background: Flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection will provide a reliable and affordable immunoassay for the rapid serosurveillance against COVID-19. Method: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture IgG immunoglobulins, which were then detected with AuNP anti-human IgG. A total of 181 samples were characterized with in-house and commercial immunoassay. The positive panel consisted of RT-PCR positive samples from patients with both <14 days and >14 days from the onset of symptoms, while the negative panel contained samples collected either from the pre-pandemic era dengue patients from healthy donors during the pandemic period. Results: In-house ELISA selected a total of 79 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7% which increased to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 97.6% and 99%. Moreover, comparative analysis between ELISA and FT-DBA revealed clinical agreement of Cohen’s Kappa value of 0.944. Conclusion: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response.

BACKGROUND:In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation.AIM:To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples.METHODS:In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined.RESULTS:The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases.CONCLUSION:The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Background Dynamics and persistence of neutralizing and non-neutralizing antibodies can give us the knowledge required for serodiagnosis, disease management, and successful vaccine design and development. The disappearance of antibodies, absence of humoral immunity activation, and sporadic reinfection cases emphasize the importance of longitudinal antibody dynamics against variable structural antigens. Methods In this study, twenty-five healthy subjects working in a SARS-COV-2 serodiagnostic assay development project were enrolled, and their sign and symptoms were followed up to six months. Three subjects showed COVID-19-like symptoms, and three subjects’ antibody dynamics were followed over 120 days by analyzing 516 samples. We have developed 12 different types of in-house ELISAs to observe the kinetics of IgG, IgM, and IgA against four SARS-CoV-2 proteins, namely nucleocapsid, RBD, S1, and whole spike (S1+S2). For the development of these assays, 30–104 pre-pandemic samples were taken as negative controls and 83 RT-qPCR positive samples as positive ones. Results All three subjects presented COVID-19-like symptoms twice, with mild symptoms in the first episode were severe in the second, and RT-qPCR confirmed the latter. The initial episode did not culminate with any significant antibody development, while a multifold increase in IgG antibodies characterized the second episode. Interestingly, IgG antibody development concurrent with IgM and IgA and persisted, whereas the latter two weans off rather quickly if appeared. Conclusion Antibody kinetics observed in this study can provide a pathway to the successful development of sero-diagnostics and epidemiologists to predict the fate of vaccination currently in place.

Detection of small amounts of target molecules with high sensitivity is important for the diagnosis of many diseases, including cancers, and is particularly important to detect early stages of disease. Here, we report the development of a temperature-responsive fusion protein (ELP-DCN) comprised of an elastin-like polypeptide (ELP), poly-aspartic acid (D), antibody-binding domain C (C), and NanoLuc luciferase (N). ELP-DCN proteins form nanoparticles above a certain threshold temperature that display an antibody-binding domain and NanoLuc luciferase on their surface. ELP-DCN nanoparticles can be applied for enhancement of immunoassay systems because they provide more antibody-binding sites and an increased number of luciferase molecules, resulting in an increase in assay signal. Here, we report the detection of human serum albumin (HSA) as a model protein using anti-HSA and ELP-DCN proteins. Upon formation of ELP-DCN nanoparticles, the detection limit improved tenfold compared to the monomeric form of ELP-DCN.

BACKGROUND:Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use. METHODS:SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD). RESULTS:In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits. CONCLUSION:The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.

Control of the cross-linking reaction is imperative when developing a sophisticated in situ forming hydrogel in the body. In this study, a heteroarmed thermoresponsive (TR) nanoparticle was designed to investigate the mechanism of controlling reactivity of the functional groups introduced into the nanoparticles. The coupling reaction was suppressed/proceeded by utilizing temperature-induced morphological changes of the TR polymer. The heteroarmed TR nanoparticle was prepared by the coassembly of amphiphilic block copolymers possessing both a TR segment and hydrophilic segment with reactive functional groups of succinimide. The longer TR chain on the nanoparticle covered the succinimide group and suppressed the reaction with the primary amine on the external nanoparticle. In contrast, the coupling reaction was promoted at a high temperature to create the chemical cross-linking structure between the nanoparticles because of the exposure of the succinimide group on the surface of the particle as a consequence of the morphological change of the TR polymer. In addition, the thermally controlled chemical reaction modulated initiation of the gelation using a highly concentrated nanoparticle solution. The heteroarmed TR nanoparticle offers great practical advantages for clinical uses, such as embolization agents, through precise control of the reaction.