Erratum: Luan, E.X.; Shoman, H.; Ratner, D.M.; Cheung, K.C.; Chrostowski, L. Silicon Photonic Biosensors Using Label-Free Detection. Sensors 2018, 18, 3519.

The authors wish to make the following corrections in their published paper in Sensors [...].

Silicon Photonic Biosensors Using Label-Free Detection.

Thanks to advanced semiconductor microfabrication technology, chip-scale integration and miniaturization of lab-on-a-chip components, silicon-based optical biosensors have made significant progress for the purpose of point-of-care diagnosis. In this review, we provide an overview of the state-of-the-art in evanescent field biosensing technologies including interferometer, microcavity, photonic crystal, and Bragg grating waveguide-based sensors. Their sensing mechanisms and sensor performances, as well as real biomarkers for label-free detection, are exhibited and compared. We also review the development of chip-level integration for lab-on-a-chip photonic sensing platforms, which consist of the optical sensing device, flow delivery system, optical input and readout equipment. At last, some advanced system-level complementary metal-oxide semiconductor (CMOS) chip packaging examples are presented, indicating the commercialization potential for the low cost, high yield, portable biosensing platform leveraging CMOS processes.

Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy against Aerosolized Intracellular Alveolar Infections.

Lung-based intracellular bacterial infections remain one of the most challenging infectious disease settings. For example, the current standard for treating Franciscella tularensis pneumonia (tularemia) relies on administration of oral or intravenous antibiotics that poorly achieve and sustain pulmonary drug bioavailability. Inhalable antibiotic formulations are approved and in clinical development for upper respiratory infections, but sustained drug dosing from inhaled antibiotics against alveolar intracellular infections remains a current unmet need. To provide an extended therapy against alveolar intracellular infections, we have developed a macromolecular therapeutic platform that provides sustained local delivery of ciprofloxacin with controlled dosing profiles. Synthesized using RAFT polymerization, these macromolecular prodrugs characteristically have high drug loading (16-17 wt % drug), tunable hydrolysis kinetics mediated by drug linkage chemistry (slow-releasing alkyllic vs fast-releasing phenolic esters), and, in general, represent new fully synthetic nanotherapeutics with streamlined manufacturing profiles. In aerosolized and completely lethal F.t. novicida mouse challenge models, the fast-releasing ciprofloxacin macromolecular prodrug provided high cure efficiencies (75% survival rate under therapeutic treatment), and the importance of release kinetics was demonstrated by the inactivity of the similar but slow-releasing prodrug system. Pharmacokinetics and biodistribution studies further demonstrated that the efficacious fast-releasing prodrug retained drug dosing in the lung above the MIC over a 48 h period with corresponding Cmax/MIC and AUC0-24h/MIC ratios being greater than 10 and 125, respectively; the thresholds for optimal bactericidal efficacy. These findings identify the macromolecular prodrug platform as a potential therapeutic system to better treat alveolar intracellular infections such as F. tularensis, where positive patient outcomes require tailored antibiotic pharmacokinetic and treatment profiles.

Sub-wavelength grating for enhanced ring resonator biosensor.

While silicon photonic resonant cavities have been widely investigated for biosensing applications, enhancing their sensitivity and detection limit continues to be an area of active research. Here, we describe how to engineer the effective refractive index and mode profile of a silicon-on-insulator (SOI) waveguide using sub-wavelength gratings (SWG) and report on its observed performance as a biosensor. We designed a 30 µm diameter SWG ring resonator and fabricated it using Ebeam lithography. Its characterization resulted in a quality factor, Q, of 7 · 103, bulk sensitivity Sb = 490 nm/RIU, and system limit of detection sLoD = 2 · 10-6 RIU. Finally we employ a model biological sandwich assay to demonstrate its utility for biosensing applications.

Nanostructured glycopolymer augmented liposomes to elucidate carbohydrate-mediated targeting.

Carbohydrate receptors on alveolar macrophages are attractive targets for receptor-mediated delivery of nanostructured therapeutics. In this study, we employed reversible addition fragmentation chain transfer polymerization to synthesize neoglycopolymers, consisting of mannose- and galactose methacrylate-based monomers copolymerized with cholesterol methacrylate for use in functional liposome studies. Glycopolymer-functional liposomes were employed to elucidate macrophage mannose receptor (CD206) and macrophage galactose-type lectin (CD301) targeting in both primary macrophage and immortal macrophage cell lines. Expression of CD206 and CD301 was observed to vary significantly between cell lines (murine alveolar macrophage, murine bone marrow-derived macrophage, RAW264.7, and MH-S), which has significant implications in in vitro targeting and uptake studies. Synthetic glycopolymers and glycopolymer augmented liposomes demonstrated specific receptor-mediated uptake in a manner dependent on carbohydrate receptor expression. These results establish a platform capable of probing endogenous carbohydrate receptor-mediated targeting via glycofunctional nanomaterials.

Performance of ultra-thin SOI-based resonators for sensing applications.

This work presents simulation and experimental results of ultra-thin optical ring resonators, having larger Evanescent Field (EF) penetration depths, and therefore larger sensitivities, as compared to conventional Silicon-on-Insulator (SOI)-based resonator sensors. Having higher sensitivities to the changes in the refractive indices of the cladding media is desirable for sensing applications, as the interactions of interest take place in this region. Using ultra-thin waveguides (<100 nm thick) shows promise to enhance sensitivity for both bulk and surface sensing, due to increased penetration of the EF into the cladding. In this work, the designs and characterization of ultra-thin resonator sensors, within the constraints of a multi-project wafer service that offers three waveguide thicknesses (90 nm, 150 nm, and 220 nm), are presented. These services typically allow efficient integration of biosensors with on-chip detectors, moving towards the implementation of lab-on-chip (LoC) systems. Also, higher temperature stability of ultra-thin resonator sensors were characterized and, in the presence of intentional environmental (temperature) fluctuations, were compared to standard transverse electric SOI-based resonator sensors.

Phage lambda capsids as tunable display nanoparticles.

Nanoparticle technologies provide a powerful tool for the development of reagents for use in both therapeutic and diagnostic, or "theragnostic" biomedical applications. Two broad classes of particles are under development, viral and synthetic systems, each with their respective strengths and limitations. Here we adapt the phage lambda system to construct modular "designer" nanoparticles that blend these two approaches. We have constructed a variety of modified "decoration" proteins that allow site-specific modification of the shell with both protein and nonproteinaceous ligands including small molecules, carbohydrates, and synthetic display ligands. We show that the chimeric proteins can be used to simultaneously decorate the shell in a tunable surface density to afford particles that are physically homogeneous and that can be manufactured to display a variety of ligands in a defined composition. These designer nanoparticles set the stage for development of lambda as a theragnostic nanoparticle system.

Identifying human milk glycans that inhibit norovirus binding using surface plasmon resonance.

Human milk glycans inhibit binding between norovirus and its host glycan receptor; such competitive inhibition by human milk glycans is associated with a reduced risk of infection. The relationship between the presence of specific structural motifs in the human milk glycan and its ability to inhibit binding by specific norovirus strains requires facile, accurate and miniaturized-binding assays. Toward this end, a high-throughput biosensor platform was developed based on surface plasmon resonance imaging (SPRi) of glycan microarrays. The SPRi was validated, and its utility was tested, by measuring binding specificities between defined human milk glycan epitopes and the capsids of two common norovirus strains, VA387 and Norwalk. Human milk oligosaccharide (HMOS)-based neoglycoconjugates, including chemically derived neoglycoproteins and oligosaccharide-glycine derivatives, were used to represent polyvalent glycoconjugates and monovalent oligosaccharides, respectively, in human milk. SPRi binding results established that the glycan motifs that bind norovirus capsids depend upon strain; VA387 capsid interacts with two neoglycoproteins, whereas Norwalk capsid binds to a different set of HMOS motifs in the form of both polyvalent neoglycoproteins and monovalent oligosaccharides. SPRi competitive binding assays further demonstrated that specific norovirus-binding glycans are able to inhibit norovirus capsid binding to their host receptors. A polyvalent neoglycoconjugate with clustered carbohydrate moieties is required for the inhibition of VA387 capsid binding to host receptor glycans, whereas both monovalent oligosaccharides and polyvalent neoglycoconjugates are able to inhibit Norwalk capsid binding to its host receptor. Binding of HMOS and HMOS-based neoglycoconjugates to norovirus capsids depends upon the specific strain characteristics, implying that HMOS and their polyvalent derivatives are potential anti-adhesive agents for norovirus prophylaxis.

Silicon photonic micro-disk resonators for label-free biosensing.

Silicon photonic biosensors are highly attractive for multiplexed Lab-on-Chip systems. Here, we characterize the sensing performance of 3 µm TE-mode and 10 µm dual TE/TM-mode silicon photonic micro-disk resonators and demonstrate their ability to detect the specific capture of biomolecules. Our experimental results show sensitivities of 26 nm/RIU and 142 nm/RIU, and quality factors of 3.3x10(4) and 1.6x10(4) for the TE and TM modes, respectively. Additionally, we show that the large disks contain both TE and TM modes with differing sensing characteristics. Finally, by serializing multiple disks on a single waveguide bus in a CMOS compatible process, we demonstrate a biosensor capable of multiplexed interrogation of biological samples.

Conformationally constrained functional peptide monolayers for the controlled display of bioactive carbohydrate ligands.

In this study, we employed thiolated peptides of the conformationally constrained, strongly helicogenic α-aminoisobutyric acid (Aib) residue to prepare self-assembled monolayers (SAMs) on gold surfaces. Electrochemistry and infrared reflection absorption spectroscopy support the formation of very well packed Aib-peptide SAMs. The immobilized peptides retain their helical structure, and the resulting SAMs are stabilized by a network of intermolecular H bonds involving the NH groups adjacent to the Au surface. Binary SAMs containing a synthetically defined glycosylated mannose-functionalized Aib-peptide as the second component display similar features, thereby providing reproducible substrates suitable for the controlled display of bioactive carbohydrate ligands. The efficiency of such Aib-based SAMs as a biomolecular recognition platform was evidenced by examining the mannose-concanavalin A interaction via surface plasmon resonance biosensing.

Giardia cyst wall protein 1 is a lectin that binds to curled fibrils of the GalNAc homopolymer.

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique beta-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWP(LRR)) and a C-terminal conserved Cys-rich region (CWP(CRR)). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (approximately 400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (approximately 1.2 microm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1(LRR). In contrast, neither MBP alone nor MBP fused to CWP1(CRR) bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.

Nanoscale clustering of carbohydrate thiols in mixed self-assembled monolayers on gold.

Self-assembled monolayers (SAMs) bearing pendant carbohydrate functionality are frequently employed to tailor glycan-specific bioactivity onto gold substrates. The resulting glycoSAMs are valuable for interrogating glycan-mediated biological interactions via surface analytical techniques, microarrays, and label-free biosensors. GlycoSAM composition can be readily modified during assembly by using mixed solutions containing thiolated species, including carbohydrates, oligo(ethylene glycol) (OEG), and other inert moieties. This intrinsic tunability of the self-assembled system is frequently used to optimize bioavailability and antibiofouling properties of the resulting SAM. However, until now, our nanoscale understanding of the behavior of these mixed glycoSAMs has lacked detail. In this study, we examined the time-dependent clustering of mixed sugar + OEG glycoSAMs on ultraflat gold substrates. Composition and surface morphologic changes in the monolayers were analyzed by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. We provide evidence that the observed clustering is consistent with a phase separation process in which surface-bound glycans self-associate to form dense glycoclusters within the monolayer. These observations have significant implications for the construction of mixed glycoSAMs for use in biosensing and glycomics applications.

Biofunctional paper via the covalent modification of cellulose.

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membrane's bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.

An organophosphonate strategy for functionalizing silicon photonic biosensors.

Silicon photonic microring resonators have established their potential for label-free and low-cost biosensing applications. However, the long-term performance of this optical sensing platform requires robust surface modification and biofunctionalization. Herein, we demonstrate a conjugation strategy based on an organophosphonate surface coating and vinyl sulfone linker to biofunctionalize silicon resonators for biomolecular sensing. To validate this method, a series of glycans, including carbohydrates and glycoconjugates, were immobilized on divinyl sulfone (DVS)/organophosphonate-modified microrings and used to characterize carbohydrate-protein and norovirus particle interactions. This biofunctional platform was able to orthogonally detect multiple specific carbohydrate-protein interactions simultaneously. Additionally, the platform was capable of reproducible binding after multiple regenerations by high-salt, high-pH, or low-pH solutions and after 1 month storage in ambient conditions. This remarkable stability and durability of the organophosphonate immobilization strategy will facilitate the application of silicon microring resonators in various sensing conditions, prolong their lifetime, and minimize the cost for storage and delivery; these characteristics are requisite for developing biosensors for point-of-care and distributed diagnostics and other biomedical applications. In addition, the platform demonstrated its ability to characterize carbohydrate-mediated host-virus interactions, providing a facile method for discovering new antiviral agents to prevent infectious disease.

A nanofiber based antiviral (TAF) prodrug delivery system.

HIV and hepatitis B are two of the most prevalent viruses globally, and despite readily available preventive treatments unforgiving treatment regimens still exist, such as daily doses of medicine that are challenging to maintain especially in poorer countries. More advanced and longer-lasting delivery vehicles can potentially overcome this problem by reducing maintenance requirements and significantly increase access to medicine. Here, we designed a technology to control the delivery of an antiviral drug over a long timeframe via a nanofiber based delivery scaffold that is both easy to produce and use. An antiviral prodrug containing tenofovir alafenamide (TAF) was synthesized by initial conjugation to glycerol monomethacrylate followed by polymerization to form a diblock copolymer (pTAF) using reversible addition-fragmentation chain transfer (RAFT). In order to generate an efficient drug delivery system this copolymer was fabricated into an electrospun nanofiber (ESF) scaffold using blend electrospinning with poly(caprolactone) (PCL) as the carrier polymer. SEM images revealed that the pTAF-PCL ESFs were uniform with an average diameter of (787 ± 0.212 nm), while XPS analysis demonstrated that the pTAF was overrepresented at the surface of the ESFs. Additionally, the pTAF exhibited a sustained release profile over a 2 month period in human serum (HS), suggesting that these types of copolymer-based drugamers can be used in conjunction with electrospinning to produce long-lasting drug delivery systems.

A versatile method for functionalizing surfaces with bioactive glycans.

Microarrays and biosensors owe their functionality to our ability to display surface-bound biomolecules with retained biological function. Versatile, stable, and facile methods for the immobilization of bioactive compounds on surfaces have expanded the application of high-throughput "omics"-scale screening of molecular interactions by nonexpert laboratories. Herein, we demonstrate the potential of simplified chemistries to fabricate a glycan microarray, utilizing divinyl sulfone (DVS)-modified surfaces for the covalent immobilization of natural and chemically derived carbohydrates, as well as glycoproteins. The bioactivity of the captured glycans was quantitatively examined by surface plasmon resonance imaging (SPRi). Composition and spectroscopic evidence of carbohydrate species on the DVS-modified surface were obtained by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. The site-selective immobilization of glycans based on relative nucleophilicity (reducing sugar vs amine- and sulfhydryl-derived saccharides) and anomeric configuration was also examined. Our results demonstrate straightforward and reproducible conjugation of a variety of functional biomolecules onto a vinyl sulfone-modified biosensor surface. The simplicity of this method will have a significant impact on glycomics research, as it expands the ability of nonsynthetic laboratories to rapidly construct functional glycan microarrays and quantitative biosensors.

The antiretroviral lectin cyanovirin-N targets well-known and novel targets on the surface of Entamoeba histolytica trophozoites.

Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man(5)GlcNAc(2)). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.

Suggestive evidence for Darwinian Selection against asparagine-linked glycans of Plasmodium falciparum and Toxoplasma gondii.

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc(3)Man(5)GlcNAc(2)) and the host 14-sugar precursor (Glc(3)Man(9)GlcNAc(2)) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.

A macrophage-targeted platform for extending drug dosing with polymer prodrugs for pulmonary infection prophylaxis.

Pulmonary melioidosis is a bacterial disease with high morbidity and a mortality rate that can be as high as 40% in resource-poor regions of South Asia. This disease burden is linked to the pathogen's intrinsic antibiotic resistance and protected intracellular localization in alveolar macrophages. Current treatment regimens require several antibiotics with multi-month oral and intravenous administrations that are difficult to implement in under-resourced settings. Herein, we report that a macrophage-targeted polyciprofloxacin prodrug acts as a surprisingly effective pre-exposure prophylactic in highly lethal murine models of aerosolized human pulmonary melioidosis. A single dose of the polymeric prodrug maintained high lung drug levels and targeted an intracellular depot of ciprofloxacin to the alveolar macrophage compartment that was sustained over a period of 7 days above minimal inhibitory concentrations. This intracellular pharmacokinetic profile provided complete pre-exposure protection in a BSL-3 model with an aerosolized clinical isolate of Burkholderia pseudomallei from Thailand. This total protection was achieved despite the bacteria's relative resistance to ciprofloxacin and where an equivalent dose of pulmonary-administered ciprofloxacin was ineffective. For the first time, we demonstrate that targeting the intracellular macrophage compartment with extended antibiotic dosing can achieve pre-exposure prophylaxis in a model of pulmonary melioidosis. This fully synthetic and modular therapeutic platform could be an important therapeutic approach with new or re-purposed antibiotics for melioidosis prevention and treatment, especially as portable inhalation devices in high-risk, resource-poor settings.

Mannose Conjugated Polymer Targeting P. aeruginosa Biofilms.

Biofilms are one of the most challenging obstacles in bacterial infections. By providing protection against immune responses and antibiotic therapies, biofilms enable chronic colonization and the development of antibiotic resistance. As previous clinical observations and studies have shown, traditional antibiotic therapy alone cannot effectively treat and eliminate biofilm forming infections due to the protection conferred by the biofilm. A new strategy specifically targeting biofilms must be developed. Here, we specifically target and bind to the PAO1 biofilm and elucidate the molecular mechanism behind the interaction between a glycan targeted polymer and biofilm using a continuous flow biofilm model. The incubation of biofilms with fluorescent glycan targeted polymers demonstrated strong and persistent interactions with the mannose-containing polymer even after 24 h of continuous flow. To evaluate the role of major biofilm proteins LecB and CdrA, loss of function experiments with knockout variants established the dual involvement of both proteins in mannose targeted polymer retention. These results identify a persistent and specific targeting strategy to the biofilm, emphasizing its potential value as a delivery strategy and encouraging further exploration of biofilm targeted delivery.