Andrea Dsouza

Phenotypic biosensors that measure bacterial viability and antimicrobial susceptibility are essential for rapid infectious disease diagnostics, yet their speed is fundamentally limited by the rate at which bacteria encounter reporter molecules, a transport bottleneck that has been typically addressed by complex microfluidic solutions. Here we show that this bottleneck can be overcome by engineering transport directly into the sensing material. A multifunctional ionic hydrogel matrix, co-encapsulating bacterial growth medium and the redox reporter resazurin, exploits swelling-driven convective transport to dramatically accelerate bacteria-reporter interactions without any change to assay chemistry. By systematically tailoring the hydrogel crosslinking density and optimizing the encapsulated nutrient-osmotic microenvironment, we maximize metabolic signal generation to achieve a 12-to-48-fold reduction in detection time relative to solution-phase and conventional hydrogel assays. Deployed in a standard 96-well format for urinary tract infection (UTI) diagnosis of 48 clinical samples, the platform rapidly detects infection in 15 minutes to 2 hours, achieving 95% sensitivity and 100% specificity for bacterial detection, and 100% sensitivity and 98% specificity for antimicrobial susceptibility profiling, compared to time-consuming gold-standard urine culture-based methods. Results are readable both quantitatively on a plate reader and visually as a colorimetric assay, enabling point-of-care deployment without additional instrumentation. Thus, embedding transport enhancement within the sensing matrix, represents a general and scalable design principle for accelerating interaction-limited biosensing, which has excellent scope for rapid diagnostic development.

Intrinsic and acquired resistance, along with the systemic toxicity of platinum and arsenic therapeutics, necessitate the development of alternative chemistries and delivery strategies for Pt- and As-containing drugs. Stimuli-responsive hydrogels offer dynamic physicochemical adaptability, making them highly suitable for biomedical applications. Herein, we investigate the pH-responsive mechanical and antimicrobial properties of platinum-containing arsenical hydrogels. Poly-(N,N-dimethylacrylamide-co-4-(N-acrylamido)-phenylarsonic acid), P-(DMAm_0.92-co-AsAm_0.08), P _As was cross-linked with Pt^II (from K₂PtCl₄) under varying pH conditions to form hydrogels. Spectroscopic techniques (UV-vis, FT-IR, ¹H, and ¹⁹⁵Pt NMR) revealed that arsenic acid protonation influences Pt^II-O-As^V interactions, impacting hydrogel integrity and dynamic behavior. Rheological analysis confirmed the pH-dependent mechanical properties, where increased pH strengthened metal-ligand interactions, enhancing material's stiffness. Self-healing properties were demonstrated via strain recovery upon cutting for all materials, while resilience upon stretching was enhanced for the looser network environment under acidic conditions. Swelling studies indicated better stability in neutral environments, whereas increased ionic strength contributed to additional structural integrity. SEM-EDX confirmed morphological changes as a function of pH, corroborating the presence of both As and Pt under all pH conditions. The antimicrobial potential of these hydrogels was evaluated against Gram-positive (, ) and Gram-negative (uropathogenic , K12 MG1655) bacteria, demonstrating a similarif not improvedantimicrobial profile in all cases compared to the individual components. This study advances the understanding of pH-modulated mechanical properties and antimicrobial activity of arsenic-platinum hydrogels, which are promising candidates for infection treatment or targeted drug delivery applications.

Bacterial adaptability to diverse environments drives infection, persistence, and antibiotic resistance. Although hydrogels are increasingly used to model such conditions, the factors governing hydrogel-dependent bacterial growth is complex. Here, we focus on agarose hydrogels and investigate how their material properties influence bacterial proliferation. Using two agarose types – hydroxyethyl substituted and unsubstituted – at varying concentrations, we tested four bacterial species ( E. coli, P. fluorescens, S. aureus, B. subtilis ) across five nutrient media yielding 120 conditions. Growth consistently decreased with increasing hydrogel stiffness and water loss in unsubstituted and substituted agarose hydrogels, regardless of species. Media effects were largely due to their impact on hydrogel properties rather than nutrient content. Furthermore, electrostatic repulsion between Gram positive bacteria and anionic unsubstituted agarose suppressed growth in high concentration gels. These findings demonstrate that bacterial growth in agarose systems is primarily shaped by gel mechanics and surface interactions, informing the design of infection models and antibacterial materials.

Platinum and arsenic (e.g., cisplatin, As₂O₃) have been used extensively in modern medicine due to their strong anticancer and antimicrobial activities. Here, polymeric arsenical scaffolds with varying As-functionalized acrylamide monomer (AsAm) composition are combined with Pt^II giving rise to hydrogels (P1-Pt–P4-Pt) with their properties being dependent on the AsAm content. The nature of the Pt–polymer interaction has been thoroughly investigated by ¹H and ¹⁹⁵Pt NMR spectroscopy as well as FT-IR, SEM, XPS, and potentiometric titration suggesting that cross-linking occurs primarily via coordination between oxygen atoms of the pendant arsenic acid group and Pt^II, while secondary noncovalent interactions are proposed to provide further structural integrity and stability. Importantly, the hydrogels demonstrate potential self-healing properties, while the nature of the cross-linking promotes deep penetration of water into the loosely cross-linked networks. Finally, preliminary qualitative antimicrobial evaluation conducted via disk diffusion assay indicates that P4-Pt is active against Gram-negative (uropathogenic Escherichia coli CFT073 and Escherichia coli K12MG1655) and Gram-positive (Bacillus subtilis and Staphylococcus aureus USA 300 JE2) bacterial strains. Overall, the combination of polymeric arsenical scaffolds with Pt^II results in the formation of cross-linked networks generating soft, strong, and self-healing hydrogels with tunable stiffness and elasticity and preliminary indications of antimicrobial potential.

Hydrogels are cross-linked networks of hydrophilic polymer chains with a three-dimensional structure. Owing to their unique features, the application of hydrogels for bacterial/antibacterial studies and bacterial infection management has grown in importance in recent years. This trend is likely to continue due to the rise in bacterial infections and antimicrobial resistance. By exploiting their physicochemical characteristics and inherent nature, hydrogels have been developed to achieve bacterial capture and detection, bacterial growth or elimination, antibiotic delivery, or bacterial sensing. Traditionally, the development of hydrogels for bacterial/antibacterial studies has focused on achieving a single function such as antibiotic delivery, antibacterial activity, bacterial growth, or bacterial detection. However, recent studies demonstrate the fabrication of multifunctional hydrogels, where a single hydrogel is capable of performing more than one bacterial/antibacterial function, or composite hydrogels consisting of a number of single functionalized hydrogels, which exhibit bacterial/antibacterial function synergistically. In this review, we first highlight the hydrogel features critical for bacterial studies and infection management. Then, we specifically address unique hydrogel properties, their surface/network functionalization, and their mode of action for bacterial capture, adhesion/growth, antibacterial activity, and bacterial sensing, respectively. Finally, we provide insights into different strategies for developing multifunctional hydrogels and how such systems can help tackle, manage, and understand bacterial infections and antimicrobial resistance. We also note that the strategies highlighted in this review can be adapted to other cell types and are therefore likely to find applications beyond the field of microbiology.

Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into the SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. SPECTRA-tube is demonstrated for the dry storage of sputum for tuberculosis detection. Mycobacterium smegmatis (Msm)-spiked mock sputum dried in a native Standard 17 glass fiber was stable for molecular testing after 10 day storage at 45 °C and for culture testing after 10- and 5-day storage at 37 °C and 45 °C, respectively. Compatibility with human sputum storage was demonstrated by dry storing 1.2 mL Mycobacterium bovis-spiked human sputum in a SPECTRA-tube for 5 days at room temperature. We have thus demonstrated the first workflow for dry storage of sputum followed by molecular and culture testing. Compared to existing specimen dry storage technologies, SPECTRA-tube significantly increases the volume of liquid specimens that can be transported in the dry state and enables the recovery of the entire sample in the liquid state, rendering it compatible with conventional downstream analysis methods.

Urinary tract infection (UTI) is one of the most common bacterial infections responsible for increased annual incidence of antimicrobial resistance (AMR) cases. Clinical diagnosis of UTI AMR relies heavily on conventional urine culture and antibiotic susceptibility testing (AST) which has a turnaround time of ∼3 days. Often, irrespective of the infection status, antibiotics are prescribed to patients even before the test results are available, leading to non-judicious use of antibiotics. Over the years, several technologies have been developed for the rapid detection and diagnosis of UTI AMR, however, most of them are limited to traditional microbiological techniques and large laboratory equipment that are not readily available in low-to-middle income countries (LMICs). To address these diagnostic limitations, we are developing a rapid and affordable UTI-AMR diagnostic microfluidic device that is clinical friendly aimed at improving UTI management and AMR stewardship. Our device enables the flow of a large volume of urine specimens for the capture/enrichment of uropathogenic bacteria and determination of AST via a porous membrane that is augmented with a multifunctional polymer-based material. Important objectives for the development of UTI AMR diagnostic microfluidic device are: (i) development of a multifunctional polymer-based material; and (ii) validation of UTI AMR diagnostic device. We have successfully developed a polysaccharide-based platform to (i) selectively capture uropathogenic bacteria from urine specimen by immobilizing concanavalin A (con A) lectin as bacterial capture agent on the polymer surface via chemical modification; (ii) encapsulate and release bacterial nutrient media and antibiotics for AST; and (iii) detect AST via encapsulation of bacterial growth indicator. In addition, we have also determined the development of methacrylate-based and acrylamide-based synthetic polymer-based material for our application. Further, we have demonstrated the uniform augmentation of the polysaccharide-based polymer onto porous membrane via dip-coating technique for on-chip bacterial capture/enrichment and AST in fluid (urine) flow conditions. The porous membrane is a conducting material which enables us to perform electrochemical measurements such as impedance spectroscopy that accelerates the detection process of antibiotic susceptibility. As a proof-of-concept, we have determined the capture of biosafety level I Escherichia coli expressing kanamycin resistance gene on chemically surface modified polysaccharide-based polymer containing con A and the antibiotic susceptibility of captured bacteria against different antibiotics with and without the porous membrane. We have quantitatively determined the limit of detection of E. coli on multifunctional polysaccharide-based polymer material. The utility of the UTI AMR microfluidic device in clinical settings enables clinicians to make informed decisions on the most appropriate antibiotic for treatment in less than a day. Integration of impedance spectroscopy will further accelerate the detection by significantly reducing the time of detection. Further, the device allows for off-chip analysis by retrieving the captured uropathogenic bacteria to perform high throughput sequencing for identifying AMR genetic determinants. Therefore, with the ability to selectively capture uropathogenic bacteria and determine AST in a short time, our technology has the potential to overcome some of the current limitations in UTI AMR diagnostics.

Technologies for preservation of specimens in the absence of cold chains are essential for optimum utilization of existing laboratory services in the developing world. We present a prototype called specimen transportation tube (SPECTRA-tube) for the collection, exposure-free drying, ambient transportation, and liquid state recovery of large-volume (>1 mL) specimens. Specimens introduced into SPECTRA-tube are dried in glass fiber membranes, which are critical for efficient liquid-state sample recovery by rehydration and centrifugation. Mycobacterium smegmatis (Msm) -spiked mock sputum dried in native Standard 17 glass fiber was stable for molecular testing after 10-day storage at 45°C, and for culture testing after 10- and 5-day storage at 37°C and 45°C, respectively. Compatibility with human sputum storage was demonstrated by dry storing Mycobacterium bovis -spiked pooled human sputum in SPECTRA-tube for 5 days at room temperature followed by successful qPCR detection. By significantly increasing the volume of samples that can be transported in the dry state and enabling recovery of the entire sample in liquid state, SPECTRA-tube presents a potential universal solution for the preservation and transportation of liquid specimens.