Organosilica Nanosensors for Monitoring Spatiotemporal Changes in Oxygen Levels in Bacterial Cultures.

Oxygen plays a central role in aerobic metabolism, and while many approaches have been developed to measure oxygen concentration in biological environments over time, monitoring spatiotemporal changes in dissolved oxygen levels remains challenging. To address this, we developed a ratiometric core-shell organosilica nanosensor for continuous, real-time optical monitoring of oxygen levels in biological environments. The nanosensors demonstrate good steady state characteristics (KpSV = 0.40 L/mg, R2 = 0.95) and respond reversibly to changes in oxygen concentration in buffered solutions and report similar oxygen level changes in response to bacterial cell growth (Escherichia coli) in comparison to a commercial bulk optode-based sensing film. We further demonstrated that the oxygen nanosensors could be distributed within a growing culture of E. coli and used to record oxygen levels over time and in different locations within a static culture, opening the possibility of spatiotemporal monitoring in complex biological systems.

Multiplex Microsphere PCR (mmPCR) Allows Simultaneous Gram Typing, Detection of Fungal DNA, and Antibiotic Resistance Genes.

OBJECTIVE: To show the high analytical specificity of our multiplex microsphere polymerase chain reaction (mmPCR) method, which offers the simultaneous detection of both general (eg, Gram type) and specific (eg, Pseudomonas species) clinically relevant genetic targets in a single modular multiplex reaction. MATERIALS AND METHODS: Isolated gDNA of 16S/rRNA Sanger-sequenced and Basic Local Alignment Tool-identified bacterial and fungal isolates were selectively amplified in a custom 10-plex Luminex MagPlex-TAG microsphere-based mmPCR assay. The signal/noise ratio for each reaction was calculated from flow cytometry standard data collected on a BD LSR Fortessa II flow cytometer. Data were normalized to the no-template negative control and the signal maximum. The analytical specificity of the assay was compared to single-plex SYBR chemistry quantitative PCR. RESULTS: Both general and specific primer sets were functional in the 10-plex mmPCR. The general Gram typing and pan-fungal primers correctly identified all bacterial and fungal isolates, respectively. The species-specific and antibiotic resistance-specific primers correctly identified the species- and resistance-carrying isolates, respectively. Low-level cross-reactive signals were present in some reactions with high signal/noise primer ratios. CONCLUSION: We found that mmPCR can simultaneously detect specific and general clinically relevant genetic targets in multiplex. These results serve as a proof-of-concept advance that highlights the potential of high multiplex mmPCR diagnostics in clinical practice. Further development of specimen-specific DNA extraction techniques is required for sensitivity testing.

Column Agglutination Assay Using Polystyrene Microbeads for Rapid Detection of Antibodies against SARS-CoV-2.

Rapid serology platforms are essential in disease pandemics for a variety of applications, including epidemiological surveillance, contact tracing, vaccination monitoring, and primary diagnosis in resource-limited areas. Laboratory-based enzyme-linked immunosorbent assay (ELISA) platforms are inherently multistep processes that require trained personnel and are of relatively limited throughput. As an alternative, agglutination-based systems have been developed; however, they rely on donor red blood cells and are not yet available for high-throughput screening. Column agglutination tests are a mainstay of pretransfusion blood typing and can be performed at a range of scales, ranging from manual through to fully automated testing. Here, we describe a column agglutination test using colored microbeads coated with recombinant SARS-CoV-2 spike protein that agglutinates when incubated with serum samples collected from patients recently infected with SARS-CoV-2. After confirming specific agglutination, we optimized centrifugal force and time to distinguish samples from uninfected vs SARS-CoV-2-infected individuals and then showed concordant results against ELISA for 22 clinical samples, and also a set of serial bleeds from one donor at days 6-10 postinfection. Our study demonstrates the use of a simple, scalable, and rapid diagnostic platform that can be tailored to detect antibodies raised against SARS-CoV-2 and can be easily integrated with established laboratory frameworks worldwide.

Wash-free paper diagnostics for the rapid detection of blood type antibodies.

Identification of specific antibodies in patient plasma is an essential part of many diagnostic procedures and is critical for safe blood transfusion. Current techniques require laboratory infrastructure and long turnaround times which limits access to those nearby tertiary healthcare providers. Addressing this challenge, a novel and rapid paper-based antibody test is reported. We validate antibody detection with reverse blood typing using IgM antibodies and then generalise the validity by adapting to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. Reagent red blood cells (RBC) are first combined with the patient plasma containing the screened antibody and a droplet of the mixture is then deposited onto paper. The light intensity profile is analyzed to identify test results, which can be detected by eye and/or with image processing to allow full automation. The efficacy of this test to perform reverse blood typing is demonstrated and the performance and sensitivity of this test using different paper types and RBC reagents was investigated using clinical samples. As an example of the flexibility of this approach, we labeled the RBC reagent with an antibody-peptide conjugate to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. This concept could be generalized to any agglutination-based antibody diagnostics with blood plasma.

Review: Nanomaterials for Reactive Oxygen Species Detection and Monitoring in Biological Environments.

Reactive oxygen species (ROS) and dissolved oxygen play key roles across many biological processes, and fluorescent stains and dyes are the primary tools used to quantify these species in vitro. However, spatio-temporal monitoring of ROS and dissolved oxygen in biological systems are challenging due to issues including poor photostability, lack of reversibility, and rapid off-site diffusion. In particular, ROS monitoring is hindered by the short lifetime of ROS molecules and their low abundance. The combination of nanomaterials and fluorescent detection has led to new opportunities for development of imaging probes, sensors, and theranostic products, because the scaffolds lead to improved optical properties, tuneable interactions with cells and media, and ratiometric sensing robust to environmental drift. In this review, we aim to critically assess and highlight recent development in nanosensors and nanomaterials used for the detection of oxygen and ROS in biological systems, and their future potential use as diagnosis tools.

Stability and Performance Study of Fluorescent Organosilica pH Nanosensors.

Long-term stability and function are key challenges for optical nanosensors operating in complex biological environments. While much focus is rightly placed on issues related to specificity, sensitivity, reversibility, and response time, many nanosensors are not capable of transducing accurate results over prolonged time periods. Sensors could fail over time due to the degradation of scaffold material, degradation of signaling dyes and components, or a combination of both. It is critical to investigate how such degradative processes affect sensor output, as the consequences could be severe. Herein, we used fluorescent core-shell organosilica pH nanosensors as a model system, incubating them in a range of common aqueous solutions over time at different temperatures, and then searched for changes in fluorescence signal, particle size, and evidence of silica degradation. We found that these ratiometric nanosensors produced stable optical signals after aging for 30 days at 37 °C in standard saline buffers with and without 10% fetal bovine serum, and without any evidence of material degradation. Next, we evaluated their performance as real-time pH nanosensors in bacterial suspension cultures, observing a close agreement with a pH electrode for control nanosensors, yet observing obvious deviations in signal based on the aging conditions. The results show that while the organosilica scaffold does not degrade appreciably over time, careful selection of dyes and further systematic investigations into the effects of salt and protein levels are required to realize long-term stable nanosensors.

Recombinant Antibody Engineering Enables Reversible Binding for Continuous Protein Biosensing.

Engineering antibodies to improve target specificity, reduce detection limits, or introduce novel functionality is an important research area for biosensor development. While various affinity biosensors have been developed to generate an output signal upon varying analyte concentrations, reversible and continuous protein monitoring in complex biological samples remains challenging. Herein, we explore the concept of directed evolution to modulate dissociation kinetics of a high affinity anti-epidermal growth factor receptor (EGFR) single-chain variable antibody fragment (scFv) to enable continuous protein sensing in a label-free binding assay. A mutant scFv library was generated from the wild type (WT) fragment via targeted permutation of four residues in the antibody-antigen-binding interface. A single round of phage display biopanning complemented with high-throughput screening methods then permitted isolation of a specific binder with fast reaction kinetics. We were able to obtain ∼30 times faster dissociation rates when compared to the WT without appreciably affecting overall affinity and specificity by targeting a single paratope that is known to contribute to the binding interaction. Suitability of a resulting mutant fragment to sense varying antigen concentrations in continuous mode was demonstrated in a modified label-free binding assay, achieving low nanomolar detection limits (KD = 8.39 nM). We also confirmed these results using an independent detection mechanism developed previously by our group, incorporating a polarity-dependent fluorescent dye into the scFv and reading out EGFR binding based on fluorescence wavelength shifts. In future, this generic approach could be employed to generate improved or novel binders for proteins of interest, ready for deployment in a broad range of assay platforms.

Rapid Gel Card Agglutination Assays for Serological Analysis Following SARS-CoV-2 Infection in Humans.

High-throughput and rapid serology assays to detect the antibody response specific to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in human blood samples are urgently required to improve our understanding of the effects of COVID-19 across the world. Short-term applications include rapid case identification and contact tracing to limit viral spread, while population screening to determine the extent of viral infection across communities is a longer-term need. Assays developed to address these needs should match the ASSURED criteria. We have identified agglutination tests based on the commonly employed blood typing methods as a viable option. These blood typing tests are employed in hospitals worldwide, are high-throughput, fast (10-30 min), and automated in most cases. Herein, we describe the application of agglutination assays to SARS-CoV-2 serology testing by combining column agglutination testing with peptide-antibody bioconjugates, which facilitate red cell cross-linking only in the presence of plasma containing antibodies against SARS-CoV-2. This simple, rapid, and easily scalable approach has immediate application in SARS-CoV-2 serological testing and is a useful platform for assay development beyond the COVID-19 pandemic.

Dynamic Solid-State Ultrasound Contrast Agent for Monitoring pH Fluctuations In Vivo.

The key challenge for in vivo biosensing is to design biomarker-responsive contrast agents that can be readily detected and monitored by broadly available biomedical imaging modalities. While a range of biosensors have been designed for optical, photoacoustic, and magnetic resonance imaging (MRI) modalities, technical challenges have hindered the development of ultrasound biosensors, even though ultrasound is widely available, portable, safe, and capable of both surface and deep tissue imaging. Typically, contrast-enhanced ultrasound imaging is generated by gas-filled microbubbles. However, they suffer from short imaging times because of the diffusion of the gas into the surrounding media. This demands an alternate approach to generate nanosensors that reveal pH-specific changes in ultrasound contrast in biological environments. Silica cores were coated with pH-responsive poly(methacrylic acid) (PMASH) in a layer-by-layer (LbL) approach and subsequently covered in a porous organosilica shell. Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were employed to monitor the successful fabrication of multilayered particles and prove the pH-dependent shrinkage/swelling of the PMASH layer. This demonstrates that reduction in pH below healthy physiological levels resulted in significant increases in ultrasound contrast, in gel phantoms, mouse cadaver tissue, and live mice. The future of such materials could be developed into a platform of biomarker-responsive ultrasound contrast agents for clinical applications.

Efficient Delivery of Dengue Virus Subunit Vaccines to the Skin by Microprojection Arrays.

Dengue virus is the most important arbovirus impacting global human health, with an estimated 390 million infections annually, and over half the world's population at risk of infection. While significant efforts have been made to develop effective vaccines to mitigate this threat, the task has proven extremely challenging, with new approaches continually being sought. The majority of protective, neutralizing antibodies induced during infection are targeted by the envelope (E) protein, making it an ideal candidate for a subunit vaccine approach. Using truncated, recombinant, secreted E proteins (sE) of all 4 dengue virus serotypes, we have assessed their immunogenicity and protective efficacy in mice, with or without Quil-A as an adjuvant, and delivered via micropatch array (MPA) to the skin in comparison with more traditional routes of immunization. The micropatch contains an ultra-high density array (21,000/cm2) of 110 µm microprojections. Mice received 3 doses of 1 µg (nanopatch, intradermal, subcutaneous, or intra muscular injection) or 10 µg (intradermal, subcutaneous, or intra muscular injection) of tetravalent sE spaced 4 weeks apart. When adjuvanted with Quil-A, tetravalent sE vaccination delivered via MPA resulted in earlier induction of virus-neutralizing IgG antibodies for all four serotypes when compared with all of the other vaccination routes. Using the infectious dengue virus AG129 mouse infectious dengue model, these neutralizing antibodies protected all mice from lethal dengue virus type 2 D220 challenge, with protected animals showing no signs of disease or circulating virus. If these results can be translated to humans, MPA-delivered sE represents a promising approach to dengue virus vaccination.

Characterization of Key Bio-Nano Interactions between Organosilica Nanoparticles and Candida albicans.

Nanoparticle-cell interactions between silica nanomaterials and mammalian cells have been investigated extensively in the context of drug delivery, diagnostics, and imaging. While there are also opportunities for applications in infectious disease, the interactions of silica nanoparticles with pathogenic microbes are relatively underexplored. To bridge this knowledge gap, here, we investigate the effects of organosilica nanoparticles of different sizes, concentrations, and surface coatings on surface association and viability of the major human fungal pathogen Candida albicans. We show that uncoated and PEGylated organosilica nanoparticles associate with C. albicans in a size and concentration-dependent manner, but on their own, do not elicit antifungal activity. The particles are also shown to associate with human white blood cells, in a similar trend as observed with C. albicans, and remain noncytotoxic toward neutrophils. Smaller particles are shown to have low association with C. albicans in comparison to other sized particles and their association with blood cells was also observed to be minimal. We further demonstrate that by chemically immobilizing the clinically important echinocandin class antifungal drug, caspofungin, to PEGylated nanoparticles, the cell-material interaction changes from benign to antifungal, inhibiting C. albicans growth when provided in high local concentration on a surface. Our study provides the foundation for defining how organosilica particles could be tailored for clinical applications against C. albicans. Possible future developments include designing biomaterials that could detect, prevent, or treat bloodstream C. albicans infections, which at present have very high patient mortality.

Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Antibody-Binding, Antifouling Surface Coatings Based on Recombinant Expression of Zwitterionic EK Peptides.

Development of antifouling films which selectively capture or target proteins of interest is essential for controlling interactions at the "bio/nano" interface. However, in order to synthesize biofunctional films from synthetic polymers that incorporate chemical "motifs" for surface immobilization, antifouling, and oriented biomolecule attachment, multiple reaction steps need to be carried out at the solid/liquid interface. EKx is a zwitterionic peptide that has previously been shown to have excellent antifouling properties. In this study, we recombinantly expressed EKx peptides and genetically encoded both surface attachment and antibody-binding motifs, before characterizing the resultant biopolymers by traditional methods. These peptides were then immobilized to organosilica nanoparticles for binding IgG, and subsequently capturing dengue NS1 as a model antigen from serum-containing solution. We found that a mixed layer of a short peptide (4.9 kDa) "backfilled" with a longer peptide terminated with an IgG-binding Z-domain (18 kDa) demonstrated selective capture of dengue NS1 protein down to ∼10 ng mL-1 in either PBS or 20% serum.

Minimum information reporting in bio-nano experimental literature.

Studying the interactions between nanoengineered materials and biological systems plays a vital role in the development of biological applications of nanotechnology and the improvement of our fundamental understanding of the bio-nano interface. A significant barrier to progress in this multidisciplinary area is the variability of published literature with regards to characterizations performed and experimental details reported. Here, we suggest a 'minimum information standard' for experimental literature investigating bio-nano interactions. This standard consists of specific components to be reported, divided into three categories: material characterization, biological characterization and details of experimental protocols. Our intention is for these proposed standards to improve reproducibility, increase quantitative comparisons of bio-nano materials, and facilitate meta analyses and in silico modelling.

Modified Organosilica Core-Shell Nanoparticles for Stable pH Sensing in Biological Solutions.

Continuous monitoring using nanoparticle-based sensors has been successfully employed in complex biological systems, yet the sensors still suffer from poor long-term stability partially because of the scaffold materials chosen to date. Organosilica core-shell nanoparticles containing a mixture of covalently incorporated pH-sensitive (shell) and pH-insensitive (core) fluorophores is presented as a continuous pH sensor for application in biological media. In contrast to previous studies focusing on similar materials, we sought to investigate the sensor characteristics (dynamic range, sensitivity, response time, stability) as a function of material properties. The ratio of the fluorescence intensities at specific wavelengths was found to be highly sensitive to pH over a physiologically relevant range (4.5-8) with a response time of <100 ms, significantly faster than that of previously reported response times using silica-based particles. Particles produced stable, pH-specific signals when stored at room temperature for more than 80 days. Finally, we demonstrated that the nanosensors successfully monitored the pH of a bacterial culture over 15 h and that pH changes in the skin of mouse cadavers could also be observed via in vivo fluorescence imaging following subcutaneous injection. The understanding gained from linking sensor characteristics and material properties will inform the next generation of optical nanosensors for continuous-monitoring applications.

Rapid and selective sampling of IgG from skin in less than 1 min using a high surface area wearable immunoassay patch.

Microprojection array (MPA) patches are an attractive approach to selectively capture circulating proteins from the skin with minimal invasiveness for diagnostics at the point-of-care or in the home. A key challenge to develop this technology is to extract sufficient quantities of specific proteins from within the skin to enable high diagnostic sensitivity within a convenient amount of time. To achieve this, we investigated the effect of MPA geometry (i.e. projection density, length and array size) on protein capture. We hypothesised that the penetrated surface area of MPAs is a major determinant of protein capture however it was not known if simultaneously increasing projection density, length and array size is possible without adversely affecting penetration and/or tolerability. We show that increasing the projection density (5000-30,000 proj. cm-2) and array size (4-36 mm2) significantly increases biomarker capture whilst maintaining of a similar level tolerability, which supports previous literature for projection length (40-190 µm). Ultimately, we designed a high surface area MPA (30,000 proj. cm-2, 36 mm2, 140 µm) with a 4.5-fold increase in penetrated surface area compared to our standard MPA design (20,408 proj. cm-2, 16 mm2, 100 µm). The high surface area MPA captured antigen-specific IgG from mice in 30 s with 100% diagnostic sensitivity compared with 10-30 min for previous MPA immunoassay patches, which is over an order of magnitude reduction in wear time. This demonstrates for the first time that MPAs may be used for ultra-rapid (<1 min) protein capture from skin in a time competitive with standard clinical procedures like the needle and lancet, which has broad implications for minimally invasive and point-of-care diagnostics.

Extending Circulating Tumor DNA Analysis to Ultralow Abundance Mutations: Techniques and Challenges.

Liquid biopsies that analyze circulating tumor DNA (ctDNA) hold great promise in the guidance of clinical treatment for various cancers. However, the innate characteristics of ctDNA make it a difficult target: ctDNA is highly fragmented, and found at very low concentrations, both in absolute terms and relative to wildtype species. Clinically relevant target sequences often differ from the wildtype species by a single DNA base pair. These characteristics make analyzing mutant ctDNA a uniquely difficult process. Despite this, techniques have recently emerged for analyzing ctDNA, and have been used in pilot studies that showed promising results. These techniques each have various drawbacks, either in their analytical capabilities or in practical considerations, which restrict their application to many clinical situations. Many of the most promising potential applications of ctDNA require assay characteristics that are not currently available, and new techniques with these properties could have benefits in companion diagnostics, monitoring response to treatment and early detection. Here we review the current state of the art in ctDNA detection, with critical comparison of the analytical techniques themselves. We also examine the improvements required to expand ctDNA diagnostics to more advanced applications and discuss the most likely pathways for these improvements.

The emerging role of nanomaterials in immunological sensing - a brief review.

Nanomaterials are beginning to play an important role in the next generation of immunological assays and biosensors, with potential impacts both in research and clinical practice. In this brief review, we highlight two areas in which nanomaterials are already making new and important contributions in the past 5-10 years: firstly, in the improvement of assay and biosensor sensitivity for detection of low abundance proteins of immunological significance, and secondly, in the real-time and continuous monitoring of protein secretion from arrays of individual cells. We finish by challenging the immunology/sensing communities to work together to develop nanomaterials that can provide real-time, continuous, and sensitive molecular readouts in vivo, a lofty goal that will require significant collaborative effort.

Development of a Multiplexed Microsphere PCR for Culture-Free Detection and Gram-Typing of Bacteria in Human Blood Samples.

Bloodstream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected bloodstream infection remain centered around blood culture (highly variable timing, in the order of hours to days to become positive), and empiric use of broad-spectrum antibiotics is therefore employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g., combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives); however, current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5 h time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labeled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labeled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.