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When a dry mass of certain amphiphiles encounters water, a spectacular interfacial instability ensues: It gives rise to the formation of ensembles of fingerlike tubular protrusions called myelin figures─tens of micrometers wide and tens to hundreds of micrometers long─representing a novel class of nonequilibrium higher-order self-organization. Here, we report that when phase-separating mixtures of unsaturated lipid, cholesterol, and sphingomyelin are hydrated, the resulting myelins break symmetry and couple their compositional degrees of freedom with the extended myelinic morphology: They produce complementary, interlamellar radial gradients of concentrations of cholesterol (and sphingomyelin) and unsaturated lipid, which stands in stark contrast to interlamellar, lateral phase separation in equilibrated morphologies. Furthermore, the corresponding gradients of molecule-specific chemistries (i.e., cholesterol extraction by methyl-β-cyclodextrin and GM1 binding by cholera toxin) produce unusual morphologies comprising compositionally graded vesicles and buckled tubes. We propose that kinetic differences in the information processing of hydration characteristics of individual molecules while expending energy dictate this novel behavior of lipid mixtures undergoing hydration. Ministry of Education (MOE) The authors also acknowledge the support from the Ministry of Education Singapore (grant no. MOE2018-T2-1-025). J.C.S.H. also acknowledges the support from Singapore Centre for Environmental Life Sciences Engineering (SCELSE). W-C.S. and A.N.P. acknowledge support from the National Science Foundation (United States) through a grant from the Division of Materials Research (#1810540). A.N.P.’s contributions are also supported by a grant from the U.S. National Science Foundation (DMR2104123).

Our study is the first report on the details of tBLM formation covering the entire time frame of the process as well as different phases of the anchoring interface.

Shape-directing agents that promote anisotropic growth are frequently employed in the synthesis of gold nanorods (GNRs), a typical example of which is the surfactant cetyltrimethylammonium bromide (CTAB). Owing to their cytotoxicity, surfactant-passivated GNRs have little use in biological applications unless made biocompatible via additional downstream processing. Reported herein is the first instance of liposome-directed anisotropic growth of GNRs synthesized in the absence of surfactants. The as-synthesized phospholipid-passivated GNRs are readily biocompatible. Among the phospholipids tested, only liposomes prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC) - a cationic transfection agent employed in lipid-mediated gene transfer in vitro - were capable of exerting shape control. By modifying a previously reported photochemical synthesis method, we developed a one-pot, seedless, DOEPC-mediated thermochemical synthesis method that yielded GNRs with an average size of 80-100 nm and an average aspect ratio of ∼3.5 and whose tips shape transformed from smooth to sharp during the course of the synthesis. Further characterization of the as-synthesized phospholipid-passivated GNRs confirmed its stability, excellent biocompatibility, photothermal transduction ability, and application in plasmonic photothermal therapy which was validated via GNR-mediated photothermal ablation of cancer cells in vitro, thus making this route of synthesis attractive for biological applications. Ministry of Education (MOE) Accepted version This work was funded by the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt10/20/14 and by the Ministry of Education, Singapore, under its MOE AcRF Tier 2 Award MOE2018-T2-1-025. This work was also supported by a grant from the Department of Biotechnology (BT/HRD/NBA/38/05/2018) awarded to R.S.

E. coli and Salmonella are two of the most common bacterial pathogens involved in food- and water-borne-related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFUmL1 of E. coli in water within 6 hours by targeting the bacterias surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants. Ministry of Education (MOE) Accepted version This work was funded by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-1-025) and the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt10/20/14.

Standard detection methods for nucleic acids, an important class of diagnostic biomarkers, are often laborious and cumbersome. In need for development of facile methodologies, localized surface plasmon resonance (LSPR) assays have been widely explored for both spectroscopic and visual detection of nucleic acids. Our sensing approach is based on monitoring changes in the LSPR band due to interaction between peptide nucleic acid (PNA) and plasmonic nanoparticles (NPs) in the presence/absence of target nucleic acid. We have investigated the importance of tuning the stoichiometry of PNA to NPs to enable "naked-eye" detection of nucleic acids at clinically relevant concentration ranges. Assaying in plasma is achieved by incorporation of silver in gold NPs (AuNPs) via an alloying process. The synthesized gold/silver alloy NPs reduce nonspecific adsorption of proteinaceous interferents in plasma. Furthermore, the gold/silver alloy NPs absorb in the most sensitive cyan to green transition zone (∼500 nm) yielding highly competitive visual limits of detection (LODs). The visual LOD (calculated objectively using the ΔE algorithm) for a model microRNA (mir21) using a productive combination of stoichiometric tuning of the PNA to NP ratio and compositional tuning of the NPs in buffer and plasma extract equals 200 pM (∼250 times lower than existing reports) and 3 nM, respectively. We envision that the proposed LSPR assay based on Au0.8Ag0.2NPs offers an avenue for rapid and sensitive on-site detection of nucleic acids in complex matrixes in combination with efficient target extraction kits.

The majority of clinical corneal prostheses (KPros) adopt a core-skirt configuration. This configuration is favored owing to the optic core (generally a cylindrical, acrylic-based material, such as PMMA), that not only provides a clear window for the patients' vision, but also confers resistance to biodegradability. The surrounding skirt (typically a biological material, such as corneal tissue) allows for host tissue integration. However, due to poor biointegration between the dissimilar core and skirt materials, it results in a weak adhesion at the interface, giving rise to clinical complications, such as bacterial infections in the tissue-PMMA interface and device extrusion. Here, we physically immobilized nano-hydroxyapatite (nHAp) on a PMMA cylinder via a dip-coating technique, to create a bioactive surface that improved biointegration in vivo. We established that the nHAp coating was safe and stable in the rabbit cornea over five weeks. More importantly, we found that apoptotic, wound healing and inflammatory responses to nHAp-coated PMMA were substantially milder than to non-coated PMMA. More mature collagen, similar to the non-operated cornea, was maintained in the corneal stroma adjacent to the nHAp-coated implant edge. However, around the non-coated cylinder, an abundant new and loose connective tissue formed, similar to bone tissue response to bioinert scaffolds. As a result of superior biointegration, tissue adhesion with nHAp-coated PMMA cylinders was also significantly enhanced compared to non-coated cylinders. This study set a precedent for the future application of the nHAp coating on clinical KPros. STATEMENT OF SIGNIFICANCE: Currently, all clinical corneal prostheses utilize as-manufactured, non-surface modified PMMA optic cylinder. The bioinert cylinder, however, has poor biointegration and adhesion with the surrounding biological tissue, which can give rise to postoperative complications, such as microbial invasion in the tissue-PMMA loose interface and PMMA optic cylinder extrusion. In the current study, we showed that surface modification of the PMMA cylinder with bioactive nano-hydroxyapatite (nHAp) significantly enhanced its biointegration with corneal stromal tissue in vivo. The superior biointegration of the nHAp-coated PMMA was signified by a more attenuated corneal wound healing, inflammatory and fibrotic response, and better tissue apposition, as well as a significantly improved corneal stromal tissue adhesion when compared to the non-coated PMMA.

Poly(3-alkylthiophene) (PT)-based conjugated polyelectrolytes (CPEs) constitute an important class of responsive polymers with excellent optical properties. The electrostatic interactions between PTs and target analytes trigger complexation and concomitant conformational changes of the PT backbones that produce distinct optical responses. These conformation-induced optical responses of the PTs enable them to be utilized as reporters for detection of various analytes by employing simple UV-vis spectrophotometry or the naked eye. Numerous PTs with unique pendant groups have been synthesized to tailor their interactions with analytes such as nucleotides, ions, surfactants, proteins, and bacterial and viral pathogens. In this perspective, we discuss PT-target analyte complexation for bioanalytical applications and highlight recent advancements in point-of-care and field deployable assays. Subsequently, we highlight a few areas of critical importance for future applications of PTs as reporters, including (i) design and synthesis of specific PTs to advance the understanding of the mechanisms of interaction with target analytes, (ii) using arrays of PTs and linear discriminant analysis for selective and specific detection of target analytes, (iii) translation of conventional homogeneous solution-based assays into heterogeneous membrane-based assay formats, and finally (iv) the potential of using PT as an alternative to conjugated polymer nanoparticles and dots in bioimaging. Ministry of Education (MOE) Nanyang Technological University This work was funded by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-1-025) and the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt10/20/14.

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Profiling of advanced glycation end products (AGEs) is an emerging area of clinical significance for disease diagnosis and prognosis. Typically, concentrations of AGEs are estimated in laboratories by trained personnel using sophisticated equipment. Herein, a facile approach for colorimetric and fluorometric profiling of AGEs is reported for rapid and on-site analysis. The concentrations of AGE levels in plasma are estimated via changes in optical properties of polythiophenes (PTs) upon interaction with aptamers (Apts) in the presence and in the absence of AGEs. To validate the proposed approach, glyceraldehyde-derived AGEs (AGE class 1 [AGE1]), the biomarker associated with cardiovascular diseases and diabetes, are used as a model system. Colorimetric analysis yielded linear responses for AGE1 for clinically relevant concentration ranges between 1.5 and 300 μg/mL with a limit of detection (LOD) of ∼1.3 μg/mL. Subsequently, an approach utilizing PTs with four different pendant groups in conjunction with four different Apts is demonstrated for qualitative colorimetric profiling and for quantitative fluorometric profiling of up to four AGEs in clinical matrices. Principal component analysis (PCA) of fluorometric responses of AGE-spiked samples yielded distinct responses for the different AGEs tested. Thus, the proposed approach ascertains rapid profiling of spiked AGEs in plasma samples without the requirement of preanalytical processing and advanced instrumentation, thereby facilitating on-site diagnosis. Ministry of Education (MOE) The authors wish to acknowledge funding support from Tier 1, MOE -RG 82/12, and NITHM Exploratory Research grant M4081989.070.

Lateral flow assays (LFAs) widely deployed for on-site diagnosis have predominantly utilized antibodies as recognition molecules. Antibodies with limited thermal stability deteriorate the performance of the LFA over time. Herein, we demonstrate a stable and robust LFA by utilizing thermally stable peptide-based 12-14 kDa affimers as recognition molecules, in lieu of conventional protein-based antibodies to analyze complex samples with a significantly improved shelf life at room temperature. The model system studied here is that of interleukin-8 (IL8) biomarker for validating the efficacy of the proposed approach, using a pair of affimer probes that demonstrates dual functionality of capturing and reporting. Affimers immobilized on the test zone of LFA serve as capture probes for IL8-affimer-MB complexes. Whereas affimers conjugated with the MBs that enable extraction of IL8 from the sample matrix serve as reporters for visual detection. The MB complexes captured at the test zone resulted in brownish test bands that enable concentration-dependent detection of IL8. The assay yielded sensitive visual detection of IL8 at ng/mL levels (~ 0.1 ng/mL and 1 ng/mL in buffer and human plasma, respectively), within 20 min, using sample volumes of ~ 100 µL. Importantly, the stability of affimer-incorporated LFA improved significantly in contrast to antibody-incorporated LFA over time, even when stored at 4 °C. Therefore, the proposed affimer-based LFA in conjunction with MBs offer stable and reliable detection of biomarkers at clinically relevant concentration ranges in complicated matrices, even without requiring cold storage, hence, offering a promising avenue for on-site diagnosis in resource-limited settings. Nanyang Technological University The Institute for Nanomedicine jointly established between Northwestern University and Nanyang Technological University provided fnancial support

Urinalysis enables non-invasive point-of-care (POC) testing of numerous biomarkers at their physiological and elevated levels, obviating the need for sophisticated equipment or trained personnel. POC urinalysis is used to identify biomarkers that are rich in urine (greater than 1 μM), such as lactate, uric acid, glucose, ions, and adenosine. Urine also contains biomarkers such as small molecules, nucleic acids, neurotransmitters, and drugs in trace amounts (less than 1 μM). These biomarkers are of significant importance for health care monitoring, diagnosis of various disorders (cancer, metabolic diseases, etc.) and illicit drug control (cocaine, steroids, etc.). While POC detection of urinary biomarkers at higher concentration (μM to mM) levels is feasible, direct assaying of biomarkers in nM to fM levels is challenging, as assay responses are typically masked by interferences from the urine sample matrix. This report is a consolidated review of emerging trends and challenges in the POC urinalysis for detecting biomarkers that are less abundant in urine. The sensing mechanisms, analytical device fabrication, discrete and integrated sample pre-treatment procedures for POC assaying of urinary markers in trace amounts are elaborated. Subsequently, the utilization of smart data analytics for facilitating personalized urinalysis is presented. A comprehensive outlook on associated challenges in POC urinalysis of biomarkers in trace amounts is further provided, which would facilitate the advancement of POC urinalysis for a wide range of healthcare applications.

In this work, we present a self-powered and on-demand transdermal drug delivery system driven by triboelectric nanogenerator (TENG). A miniaturized TENG and a home-built power management circuit were designed to trigger the electric-responsive drug carrier for controlled drug release, as well as to activate the iontophoresis treatment for enhanced drug delivery efficiency. In the system, the TENG can harvest electricity from bio-mechanical energy, and the power management circuit is able to store, adjust, and stabilize the electricity for on-demand drug release actions. Our results demonstrate that the on-demand drug release can be simply realized by operating the TENG. Manually rotating the TENG (30–40 rpm) for 1.5 min can release a drug dosage of 3 μg/cm2. Furthermore, the system has achieved tunable drug release rate for the transdermal drug delivery: the rate can be tuned from 0.05 to 0.25 μg/cm2 per minute by changing the duration of TENG charging or the resistance of the power management circuit. In addition, ex vivo experiments on porcine skin validate the performance of such TENG-based drug delivery system with ∼50% enhancement over conventional transdermal patches. The proposed system is intended to provide patients with an easy approach to achieve customized rate and dosage of drug release.

A flow-through colorimetric assay for detection of nucleic acids in plasma is reported. The proposed assay features an array of four polyvinylidene fluoride (PVDF) membranes impregnated with cationic poly (3-alkoxy-4-methylthiophene) (PT) as an optical reporter. The sensing strategy is based on monitoring the changes in optical properties of PT, upon complexation with target nucleic acids in the presence and in the absence of their corresponding complementary peptide nucleic acids (PNAs). As a proof of concept, the proposed methodology is validated using two biomarkers; lung cancer associated microRNA (mir21) and hepatitis B virus DNA (HBV-DNA). The flow-through colorimetric assay enabled detection of mir21 and HBV-DNA in plasma without requiring tedious sample pre-treatment and clean up protocols. Colorimetric responses for mir21 and HBV-DNA were obtained at nanomolar concentrations over five orders of magnitudes (from 1 nM to 10 μM), with a limit of detection of ∼0.6 nM and ∼2 nM in DI water and plasma, respectively. A logic gate system was developed to utilize the colorimetric assay responses as inputs for discrimination of mir21 and HBV-DNA and subsequently to obtain a profile of nucleic acids in samples that exceed respective clinical threshold limits, thereby enabling rapid and point of care (POC) disease diagnosis. Furthermore, the proposed methodology can be utilized for detection of a large number of nucleic acids in plasma by extending the array of PT impregnated membranes incorporated with their corresponding complementary PNAs. Accepted version

Lateral flow assays (LFA) have been extensively explored for rapid and cost effective point-of-care diagnostics. Typically, LFA responses are influenced by the complexity of sample matrices containing analogues or molecules that may potentially yield non-specific responses, for instance, when assaying for biomarkers in blood, serum and plasma. Therefore, isolation of analytes of interest from sample matrices would significantly improve the LFA responses. Herein, we report a magnetic field assisted preconcentration approach for extraction and assay of proteins in an LFA format. Cardiac marker, Troponin (cTnICT complex), is utilized as a model system for validation of the proposed approach. Magnetic fields of different strengths are evaluated for retaining cTnICT on an LFA membrane utilizing magnetic beads conjugated with anti-Troponin I (anti-TnI) antibodies. The sample matrix components subsequently flows through to the absorbent pad via a hydrophilic passivation layer that is protecting the capturing anti-Troponin C (anti-TnC) antibodies in the test zone. Isolated TnI-magnetic bead complexes are then released to flow downstream along the LFA strip to the test zone upon removal of magnetic field and the passivation layer. The capture of cTnICT magnetic bead complexes at the test zone produces a characteristic brownish band, enabling concentration dependant visual detection of cTnICT. Experimental results indicate that the assay yields pM level sensitivity within 15 min using very low sample volumes (

This paper describes an approach for colorimetric detection of salivary α-amylase, one of the potential biomarkers of autonomic nervous system (ANS) activity, for enabling assessment of fatigue. The ability of α-amylase to cleave α-bonds of polysaccharides is utilized for developing a colorimetric assay. In the proposed approach, 2-chloro-4-nitrophenyl-α-d-maltotrioside as substrate releases a colored byproduct upon cleavage by salivary α-amylase. Introduction of maltose as a noncompetitive inhibitor yields desirable linear responses in the physiologically relevant concentration range (20–500 μg/mL) with a limit of detection (LOD) of 8 μg/mL (in aqueous solution). The concentrations of substrate and noncompetitive inhibitor are subsequently optimized for colorimetric detection of salivary α-amylase. A facile paper-based “strip” assay is proposed for analysis of human saliva samples with marginal interference from saliva components. The proposed assay is rapid, specific, and easy-to-implement for colorimetric detection of salivary α-amylase between 20 and 500 μg/mL. Complementary RGB (red, green, blue components) analysis offers quantitative detection with a LOD of 11 μg/mL. The two assay formats are benchmarked against the Phadebas test, a state of the art method for spectrophotometric detection of α-amylase. The reported paper-based methodology possesses a high potential for estimation of altered ANS responses toward stressors that possibly could find applications in assessment of fatigue and for monitoring onset of fatigue. Nanyang Technological University Accepted version This work is supported by Provost’s office and Institute for Sports Research (ISR), School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore.

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation. Accepted version

The extremely toxic botulinum neurotoxin poses a threat for health and food safety, requiring rapid and easy-to-use detection platforms. To meet these requirements, we have explored a novel functional assay format for detection of botulinum neurotoxin serotype A light chain (BoNT/A LC) in complex matrices. The proposed assay utilizes a synthetic peptide designed to mimic the SNAP-25 protein (synaptosomal-associated protein 25) as substrate bound to a superparamagnetic bead and a fluorescent dye. Cleavage of the peptide by BoNT/A LC yields a reduction in fluorescence signal revealing the presence of the BoNT/A LC in the sample matrices tested. The superparamagnetic beads enable efficient separation of the cleaved peptides from food matrices, thereby improving the reliability of responses. Herein, we demonstrate a protocol offering an assay time of 6 h and a LOD of 0.5 nM (25 ng/ml). The proposed protocol is validated using carrot juice and diluted milk pending further improvements in sensitivity and overall assay time. Robustness, cost-effectiveness, low sample volume requirements in conjunction with the possibility of multiplexing for other proteolytic enzymes makes the proposed protocol competitive in comparison with conventional BoNT assays reported elsewhere.

Bacterial infection following implantation of an artificial corneal scaffold is a serious complication. Conventional antibiotic prophylaxis, which includes topical vancomycin application, is limited by low bioavailability, high dosing requirement, and poor patient compliance. The ideal option to overcome these issues is an antibiotic-eluting corneal prosthesis that sustains the local release of drug. In this study, we incorporated vancomycin in thick 15% collagen hydrogels to create an artificial corneal scaffold with anti-infective capability. The incorporation of vancomycin did not significantly alter the Young's modulus, transparency and refractive index of the vancomycin-loaded hydrogel (VH), which were 0.79 ± 0.04 MPa (p = 0.233 compared to blank hydrogel), 94.3 ± 2.3% (p = 0.115) and 1.346 ± 0.005 (p = 0.264), respectively. In vitro, the drug elution was sustained for up to 7 days. The VH was subsequently implanted intrastromally in rabbit corneas, replacing stromal tissue that was removed following femtosecond laser-assisted small incision lenticule extraction procedure. In vivo, the vancomcyin could be detected in the aqueous humor for up to 10 days. We then created a corneal infectious keratitis model by intrastromal injection of 1 × 108 CFU/ml of Staphylococcus aureus inoculate on day 2 postimplantation. On day 3 postinfection, the VH-implanted corneas were clear and nonedematous and showed a substantial reduction of log 2.5 in S. aureus compared to the blank hydrogel-implanted corneas, which appeared hazy, edematous, and had excessive inflammation. Immunohistochemistry of inflammatory marker, CD18, demonstrated a significant reduction in inflammatory cells in VH-implanted corneas (49 ± 9 cells/unit area) compared to blank hydrogel-implanted corneas (523 ± 15 cells/unit area) (p < 0.001). In conclusion, we have demonstrated the efficacy of a drug-eluting corneal implant in preventing perioperative bacterial infections.

Polyampholytic poly(2-aminoethyl methacrylate-co-sulfopropyl methacrylate) (p(AEMA-co-SPMA)) thin films were prepared by self-initiated photopolymerization and photografting (SIPGP) and are demonstrated to be a potential alternative to films prepared from zwitterionic poly(sulfobetaine methacrylate) (pSBMA) for antifouling applications. SIPGP allows polymerization from aqueous solutions containing only monomers, implying that p(AEMA-co-SPMA) thin films can be prepared simply and inexpensively without the risk of introducing potentially toxic substances necessary in many controlled polymerization reactions. For the polymers, wettabilities were studied by contact angle goniometry, the compositions of the films were determined by infrared and X-ray photoelectron spectroscopies, and streaming current measurements were used to assess their net charge. The antibiofouling properties were compared via adsorption of fibrinogen and bovine serum albumin, settlement of algal zoospores, and the growth of sporelings of the marine alga Ulva lactuca. The fouling of the p(AEMA-co-SPMA) copolymer was in several respects similar to that of the zwitterionic pSBMA and suggests that it is potentially suitable for applications under high-salinity conditions, such as marine or physiological environments. Funding Agencies|European Communitys Seventh Framework Program FP7/2007-2013 [237997]; Swedish Research Council (VR)Swedish Research Council [2014-04004, 2017-06696, 621-2013-5357]; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University (Faculty Grant SFO-Mat-LiU) [2009-00971]; Carl Tryggers Stiftelse [CTS 15:507]

International audience; Antibody-Gold nanoparticle (Ab-AuNP) bioconjugates are widely used in the field of biosensing. This prompted researchers to set up various strategies to conjugate antibodies to gold nanoparticles. Optimal conjugation is of critical importance, as the Ab-AuNP bioconjugates should be stable while maintaining the ability of the antibody to recognize and bind its corresponding antigen. All the same, a high coverage of antibodies on AuNPs is a key-step to build up a sensitive biosensor, but an ideal coverage requires to be perfectly balanced with the orientation and accessibility of the conjugated antibodies. In this review, we intend to provide the reader with the key elements allowing for mastering the conjugation of Ab to AuNP and rationalizing, at the molecular level, the mechanisms involved together with the expected antibody coverages and orientations. We will focus on IgG-type antibodies conjugated to spherical AuNPs as these bioconjugates are the most commonly used ones for biosensors. First, we report an exhaustive survey of the methods of conjugation, via strategies of physisorption and chemisorption. Then we provide a critical restitution of the relevant strategies allowing the quantification of antibodies coverage on gold nanoparticles either through direct analysis of the bioconjugates or indirect analysis of the supernatant. In the last part, we review and discuss selected applications of these Ab-AuNP bioconjugates in optical biosensing.

Standard detection methods for nucleic acids, an important class of diagnostic biomarkers, are often laborious and cumbersome. In need for development of facile methodologies, localized surface plasmon resonance (LSPR) assays have been widely explored for both spectroscopic and visual detection of nucleic acids. Our sensing approach is based on monitoring changes in the LSPR band due to interaction between peptide nucleic acid (PNA) and plasmonic nanoparticles (NPs) in the presence/absence of target nucleic acid. We have investigated the importance of tuning the stoichiometry of PNA to NPs to enable "naked-eye" detection of nucleic acids at clinically relevant concentration ranges. Assaying in plasma is achieved by incorporation of silver in gold NPs (AuNPs) via an alloying process. The synthesized gold/silver alloy NPs reduce nonspecific adsorption of proteinaceous interferents in plasma. Furthermore, the gold/silver alloy NPs absorb in the most sensitive cyan to green transition zone (∼500 nm) yielding highly competitive visual limits of detection (LODs). The visual LOD (calculated objectively using the Δ

Spatially controlled reactions at the nanoscale have attracted increasing interest for fundamental chemistry and for the engineering of novel functional materials. Herein, we demonstrate that pH-triggered reduction of silver ions preferentially occurs at the inner walls of porous and citrate-capped gold nanoshell (AuNS) particles. The reaction initially relies on the presence of sacrificial silver ions inside the AuNS particles as well as in the surrounding preparation solution, and it proceeds upon external addition of silver ions until a solid silver core is formed inside the AuNS particles. Subsequent reduction of silver occurs on the external surface of the solidified AuNS, resulting in a layered and compositionally complex nanoparticle containing both silver and gold. Growth experiments performed in the dark, under white light illumination, as well as near resonance suggest that the reduction reaction is not guided by a plasmonic field enhancement effect. This is in contrast to the recently proposed hot spot mechanism of silver reduction at the rim of nanoholes in a periodic gold array. Our observations point toward a confinement process that proceeds via a continuous supply of silver ions that diffuse from the external solution through the porous shell into the inner volume of the AuNS particles where they become reduced. Accepted version

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38, MW, 1.25&ndash, 3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.

Compartmentalization, as a design principle, is a prerequisite for the functioning of eukaryotic cells. Although cell mimics in the form of single vesicular compartments such as liposomes or polymersomes have been tremendously successful, investigations of the corresponding higher-order architectures, in particular bilayer-based multicompartment vesicles, have only recently gained attention. We hereby demonstrate a multicompartment cell-mimetic nanocontainer, built-up from fully synthetic membranes, which features an inner compartment equipped with a channel protein and a semi-permeable outer compartment that allows passive diffusion of small molecules. The functionality of this multicompartment architecture is demonstrated by a cascade reaction between enzymes that are segregated in separate compartments. The unique architecture of polymersomes, which combines stability with a cell-membrane-mimetic environment, and their assembly into higher-order architectures could serve as a design principle for new generation drug-delivery vehicles, biosensors, and protocell models.

Membrane-intercalating conjugated oligoelectrolytes (COEs) are emerging as potential alternatives to conventional, yet increasingly ineffective, antibiotics. Three readily accessible COEs, belonging to an unreported series containing a stilbene core, namely D4, D6, and D8, were designed and synthesized so that the hydrophobicity increases with increasing side-chain length. Decreased aqueous solubility correlates with increased uptake by E. coli. The minimum inhibitory concentration (MIC) of D8 is 4 μg mL-1 against both E. coli and E. faecalis, with an effective uptake of 72 %. In contrast, the MIC value of the shortest COE, D4, is 128 μg mL-1 owing to the low cellular uptake of 3 %. These findings demonstrate the application of rational design to generate efficacious antimicrobial COEs that have potential as low-cost antimicrobial agents.

Background An important part of water quality control is waterborne pathogen detection. Conventional microorganism detections are done by costly and complicated biochemical approaches, which leave chemical pollutions and require professional laboratories, technicians, and much time. Recently, machine-learning-based image classification algorithms are developed for faster, cheaper, simpler, and more environmentally friendly microorganism detection. Most of them are designed for conventional microscopic images. On the other hand, the optofluidic system is a smaller and cheaper alternative to conventional microscopes, so we aim to propose a microorganism classification algorithm for optofluidic images. Method In our previous study, the Binarized-Greyscale-Hybrid Algorithm with Multi-Region Binarization (BiGHAM) algorithm was developed for our optofluidic image dataset. In this paper, a new circle-based region partition method for the Multi-Region Binarization and a new mutual-information-based feature selection method were proposed to improve the BiGHAM method. Additionally, an implementation example of how to adapt and apply our method on an open dataset was presented. Results and conclusion With the circle-based region partition method and the new feature combination selected based on mutual information, the improved-BiGHAM algorithm achieved higher classification accuracies, and the algorithm is simpler than the previous BiGHAM method during training. For our optofluidic image dataset, the accuracy improvement for the 1000-image-per-class case was from 94.8 to 96.3%, and for the 30-image-per-class case was from 77.8 to 85.6% with the support vector machine as the classifier. The adapted algorithm also works well on the open dataset, showing the potential to be extended to applications on different microorganism images.

Point of care testing (POCT) platforms are analytical devices used for rapid on-site sensing applications. POCT in sports science has attracted significant attention as the markers related to physiological changes and metabolism can be analysed on-site for monitoring an athlete's health, performance, recovery and even for doping control. POCT platforms also possess the potential to facilitate trainers in optimizing the training regimes for preventing injuries, track their nutrition, and could potentially assist sports committees to enforce effective doping control measures during and outside competitions. This review is a consolidated report on emerging trends in POCT platforms in sports science. The methodologies reported thus far that have already been applied or possess a potential to be applied for detection of various sports biomarkers are elaborated. A comprehensive outlook is further provided, which would aid efforts on development of POCT platforms for a wide range of applications. Ministry of Education (MOE) Nanyang Technological University We would like to thank to MOE Tier 1 Grant, Singapore (Grant number 2018-T1-001-079) and Provost Office NTU, Singapore for supporting this work.

Identifying peptide substrates that are efficiently cleaved by proteases can give insights into the substrate recognition and specificity, guide the development of inhibitors and improve the sensitivity of assays. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a >400 fold improvement in OmpT's catalytic efficiency with a kcat/Km value of 6.1 x 106 M-1 s-1. Interestingly, wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

In designing unmodified gold nanoparticles-based aptasensing (uGA) assays for antibiotics, we find that some antibiotics can adsorb directly on gold nanoparticles (GNP) regardless of the presence of aptamers, which have been long overlooked in the past. Some adsorptions, however, would strongly disturb the charge distribution on the GNP surface, break up the static colloidal profile, and thus generate false positive colorimetric signals. To identify antibiotics qualified for uGA assays, we established two rational screening criteria for antibiotic targets relying on their oil–water partition coefficients (log P values) and net physiological charges: log P > 0 and charge ≤0. A good agreement of the GNP color change was obtained between the two criteria-based predictions and the actual tests using six representative antibiotics. The proposed criteria help to shed light on GNP–target interactions, which is significant for developing novel GNP-based colorimetric assays with high reliability.

The synthesis and characterization of a new protein-polymer conjugate composed of β lactoglobulin A (βLG A) and poly(ethylene glycol) PEG is described. βLG A was selectively modified to self-assemble by super-charging via amination or succinylation followed by conjugation with PEG. An equimolar mixture of the oppositely charged protein-polymer conjugates self-assemble into spherical capsules of 80-100 nm in diameter. The self-assembly proceeds by taking simultaneous advantage of the amphiphilicity and polyelectrolyte nature of the protein-polymer conjugate. These protein-polymer capsules or proteinosomes are reminiscent of protein capsids, and are capable of encapsulating solutes in their interior. We envisage this approach to be applicable to other globular proteins.

A highly sensitive approach for rapid and label-free detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) using an optofluidic chip is demonstrated. The optofluidic chip is prepared by covalent immobilization of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to an integrated microring resonator. Subsequent detection of 2,4-D carried out in a competitive immunoreaction format enables selective detection of 2,4-D in different types of water samples, including bottled, tap, and lake water, at a limit of detection (LOD) of 4.5 pg/mL and in a quantitative range of 15-105 pg/mL. The microring resonator-based optofluidic chip is reusable with ultrahigh sensitivity that offers real-time and on-site detection of low-molecular-weight targets for potential applications in food safety and environmental monitoring. NRF (Natl Research Foundation, S’pore) Accepted version

Qingwen Zhang,1,2,* Lin Wu,3,* Ten It Wong,4 Jinling Zhang,5 Xiaohu Liu,5 Xiaodong Zhou,4 Ping Bai,3 Bo Liedberg,5 Yi Wang1,2,5 1School of Ophthalmology and Optometry, Eye Hospital, School ofBiomedical Engineering, Wenzhou Medical University, 2Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences, Wenzhou, People’s Republic of China; 3Electronics and Photonics Department, Institute of High Performance Computing, 4Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 5Centre for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore *These authors contributed equally to this work Abstract: Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay. Keywords: gold nanohole array, localized surface plasmon, propagating surface plasmon, fluorescence enhancement, prostate-specific antigen

An aqueous, room temperature, and surfactant-less synthetic route based on galvanic replacement and co-reduction is reported to yield PdAgCu nanoparticles (NPs) with a high density of sharp and branched tips. The PdAgCu NPs are of high-purity, uniform size and more than 90% of them adopt branched tips. Besides, the PdAgCu NPs exhibit hollow interiors, well-alloyed nature, and a tuneable localized surface plasmon resonance peak in the near infrared region. Their morphology and optical property are facilely controlled by adjusting the precursor molar ratio, amounts of AgNP seeds and Cl- ions in the growth solution. The proposed synthetic approach is anticipated to offer an attractive avenue for facile synthesis of other multi-metallic and branched NPs with controlled properties.

Analogues and interferents present in complex matrices like blood, plasma or serum influence lateral flow assay (LFA) responses, resulting in weak signals which compromise the limit of detection. Herein, we report a gold nanoparticle conjugated magnetic bead (GMB) based LFA approach for extraction and sensitive visual assaying of proteins in plasma. To validate the approach GMB, conjugated with anti-Troponin I antibodies, is used to capture cardiac marker Troponin I-C-T (cTnICT) in plasma that subsequently flows through on LFA membrane via a test zone containing anti-Troponin C antibodies. The capture of cTnICT-GMB complexes at the test zone produces a characteristic brownish band, enabling concentration dependent visual detection of cTnICT. Subsequently, contrast of these bands are enhanced by a nucleation approach, that utilizes the gold nanoparticles on GMB in the test bands as seed materials for growth of more visible gold clusters, improving visual detection limit of the assay to 0.1 ng/mL in plasma within 20 min, using low sample volumes (

Over the past few decades, colorimetric assays have been developed for cost-effective and rapid on-site urinalysis. Most of these assays were employed for detection of biomarkers such as glucose, uric acid, ions, and albumin that are abundant in urine at micromolar to millimolar levels. In contrast, direct assaying of urinary biomarkers such as glycated proteins, low-molecular-weight reactive oxygen species, and nucleic acids that are present at significantly lower levels (nanomolar to picomolar) remain challenging due to the interferences from the urine sample matrix. State-of-the-art assays for detection of trace amounts of urinary biomarkers typically utilize time-consuming and equipment-dependent sample pretreatment or clean-up protocols prior to assaying, which limits their applicability for on-site analysis. Herein, we report a colorimetric assay for on-site detection of trace amount of generic biomarkers in urine without involving tedious sample pretreatment protocols. The detection strategy is based on monitoring the changes in optical properties of poly(3-(4-methyl-3'-thienyloxy)propyltriethylammonium bromide) upon interacting with an aptamer or a peptide nucleic acid in the presence and absence of target biomarkers of relevance for the diagnosis of metabolic complications and diabetes. As a proof of concept, this study demonstrates facile assaying of advanced glycation end products, 8-hydroxy-2'-deoxyguanosine and hepatitis B virus DNA in urine samples at clinically relevant concentrations, with limits of detection of ∼850 pM, ∼650 pM, and ∼ 1 nM, respectively. These analytes represent three distinct classes of biomarkers: (i) glycated proteins, (ii) low-molecular-weight reactive oxygen species, and (iii) nucleic acids. Hence, the proposed methodology is applicable for rapid detection of generic biomarkers in urine, without involving sophisticated equipment and skilled personnel, thereby enabling on-site urinalysis. At the end of the contribution, we discuss the opportunity to translate the homogeneous assay into a paper-based format. Ministry of Education (MOE) Nanyang Technological University The authors wish to acknowledge funding support from Tier 1, MOE -RG 82/12, and NITHM Exploratory Research grant M4081989.070. B.O.B. is supported by an Ong Tiong Tat Chair Professorship and MOE Tier 1 project 2017-T1-001- 139.

The localized surface plasmon resonance (LSPR) phenomenon provides a versatile property for biodetection. Herein, this unique feature was employed to build a homogeneous optical biosensor to detect staphylococcal enterotoxin A (SEA) in solution down to very low levels by naked-eye readout. If the initial position of the LSPR band is located in the cyan region, even a small red shift (∼2-3 nm) induced by a refractive index change close to the surface of nanoparticles (NPs) could make the light absorption transit from cyan to green and become visually detectable via a concomitant change in the complementary colors. In this work, we aimed at synthesizing two types of NPs based on compositionally complex core-shell NPs-Ag shells on AuNPs (Au@AgNPs) and Ag inside gold nanoshells (Ag@AuNPs). By controlling the thickness of the shells and their surface chemistry with anti-SEA antibody (Ab), the LSPR band was tuned to near 495 and 520 nm for Ag@AuNPs and Au@AgNPs, respectively. The two particle systems were subsequently applied to spectroscopically and visually detect anti-SEA Ab-SEA interactions. Upon the addition of SEA, large red shifts of the LSPR band were observed spectroscopically and the limits of detection (LODs) were estimated to be 0.2 and 0.4 nM for Au@AgNPs and Ag@AuNPs, respectively. Although the two sets of NPs gave almost identical LODs, the Ag@AuNPs whose initial position of the LSPR band was tuned in the cyan to green region (∼500 nm) displayed a substantially more distinct color change from orange to red, as revealed by the naked eye. We foresee significant potential to this strategy in medical diagnostics and environmental monitoring, especially when basic laboratory infrastructure is sparse or nonexistent.

Efficient removal of interferents from complex matrices would significantly improve the performance of state of the art dipstick assays. Herein, we evaluate a graphitized carbon black (GCB)-incorporated dipstick, a configuration that has not been explored before, for reliable and facile on-site analysis of complex matrices. Carrot juice, a highly pigmented sample matrix, is chosen for evaluating the retention of interferents within the sorbent-incorporated cleanup pad on the dipstick. A peptide with a specific cleavage site for botulinum neurotoxin A light chain (BoNT/A LC), a model protease for validation of the proposed dipstick assay, is incubated with the test samples containing BoNT/A LC. Subsequently, the BoNT/A LC digested substrate and sample matrix flow vertically through the GCB-deposited cleanup pad within which the matrix interferents are captured, while the substrate, with a minimum of interferents, continues to flow toward a conjugation pad for labelling with Europium particles. Finally, the cleaved and uncleaved substrates flow toward a detection zone, where they bind to the test line producing a pinkish band which is not visible in the absence of GCB incorporation. The dipstick assay yields a LOD of 0.1 nM (5 ng/mL) of BoNT/A LC in carrot juice, within 20 min. The reported approach enables detection of proteases in a wide range of matrices upon incorporation of appropriate sorbents, ultimately aiming to exclude tedious laboratory-based sample pre-treatment protocols. Thus, merging extraction, cleanup, and pre-concentration steps with a sensitive optical detection approach is an attractive strategy for on-site assaying in complex matrices. Graphical abstract.