Antibody-functionalized aluminum oxide-coated particles targeting neutrophil receptors in a multifrequency microfluidic impedance cytometer.

Personalized diagnostics of infectious diseases require monitoring disease progression due to their ever-changing physiological conditions and the multi-faceted organ system mechanisms involved in disease pathogenesis. In such instances, the recommended clinical strategies involve multiplexing data collection from critical biomarkers related to a patient's conditions along with longitudinal frequent patient monitoring. Numerous detection technologies exist both in research and commercial settings to monitor these conditions, however, they fail to provide biomarker multiplexing ability with design and data processing simplicity. For a recently conceived multiplexing biomarker modality, this work demonstrates the use of electrically sensitive microparticles targeting and identifying membrane receptors on leukocytes using a single detection source, with a high potential for multiplexing greater than any existing impedance-based single-detection scheme. Here, polystyrene microparticles are coated with varying thicknesses of metal oxides, which generate quantifiable impedance shifts when exposed to multifrequency electric fields depending on the metal oxide thickness. Using multifrequency impedance cytometry, these particles can be measured and differentiated rapidly across one coplanar electrode scheme. After surface-functionalizing particles with antibodies targeting CD11b and CD66b receptors, the particles are combined with isolated neutrophils to measure receptor expression. A combination of data analysis techniques including multivariate analysis, supervised machine learning, and unsupervised machine learning was able to accurately differentiate samples with up to 91% accuracy. This proof-of-concept study demonstrates the potential for these oxide-coated particles for enumerating specific leukocytes enabling multiplexing. Further, additional coating thicknesses or different metal oxide materials can enable a compendium of multiplexing targeting resource to be used to develop a high-multiplexing sensor for targeting membrane receptor expression.

Salvadora persica mediated synthesis of silver nanoparticles and their antimicrobial efficacy.

Silver nanoparticles (AgNPs) exhibit strong antimicrobial properties against many pathogens. Traditionally employed chemical methods for AgNPs synthesis are toxic for the environment. Here, we report a quicker, simpler, and environmentally benign process to synthesize AgNPs by using an aqueous 'root extract' of Salvadora persica (Sp) plant as a reducing agent. The synthesized Salvadora persica nano particles (SpNPs) showed significantly higher antimicrobial efficacy compared to earlier reported studies. We characterized SpNPs using UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FE-SEM), Dynamic Light Scattering (DLS) and X-ray powder diffraction (P-XRD). UV-Vis spectrum showed the highest absorbance at 420 nm. FTIR analysis depicts presence of bond stretching including OH- (3300 cm-1), C=N- (2100 cm-1) and NH- (1630 cm-1) which are attributed in the involvement of phenolics, proteins or nitrogenous compounds in reduction and stabilization of AgNPs. TEM, FE-SEM and DLS analysis revealed the spherical and rod nature of SpNPs and an average size of particles as 37.5 nm. XRD analysis showed the presence of the cubic structure of Ag which confirmed the synthesis of silver nanoparticles. To demonstrate antimicrobial efficacy, we evaluated SpNPs antimicrobial activity against two bacterial pathogens (Escherichia coli (ATCC 11229) and Staphylococcus epidermidis (ATCC 12228)). SpNPs showed a significantly high inhibition for both pathogens and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were found to be 0.39 µg/mL and 0.78 µg/mL for E. coli while 0.19 µg/mL and 0.39 µg/mL for S. epidermidis respectively. Further, Syto 16 staining of bacterial cells provided a supplemental confirmation of the antimicrobial efficacy as the bacterial cells treated with SpNPs stop to fluoresce compared to the untreated bacterial cells. Our highly potent SpNPs will likely have a great potential for many antimicrobial applications including wound healing, water purification, air filtering and other biomedical applications.

Functionalization of hybrid surface microparticles for in vitro cellular antigen classification.

Hybrid material surfaces on microparticles are emerging as vehicles for many biomedical multiplexing applications. Functionalization of these hybrid surface microparticles to biomolecules presents unique challenges related to optimization of surface chemistries including uniformity, repeatability, and sample sparring. Hybrid interfaces between microlevel surfaces and individual biomolecules will provide different microenvironments impacting the surface functionalization optimization and efficiency. Here, we propose and validate the first demonstration of streptavidin adsorption-based antibody functionalization on unmodified, hybrid surface microparticles for in vitro analysis. We test this analytical technique and fabricate hybrid surface microparticles with a polystyrene core and aluminum oxide semi-coating. Additionally, we optimize the streptavidin-biotin functionalization chemistry in both assay implementation and sample sparring via analytical mass balances for these microparticles and subsequently conjugate anti-human CD11b antibodies. Result confirmation and characterization occurs from ultraviolet protein absorbance and ImageJ processing of fluorescence microscopy images. Additionally, we design and implement the multi-sectional imaging (MSI) approach to support functionalization uniformity on the hybrid surface microparticles. Finally, as a proof-of-concept performance, we validate anti-CD11b antibodies functionalization by visualizing hybrid surface microparticles conjugate to human neutrophils isolated from blood samples collected from potentially septic patients. Our study introduces and defines a category of functionalization for hybrid surface microparticles with the intent of minuscule sample volumes, low cost, and low environmental impact to be used for many cellular or proteomic in vitro multiplexing applications in the future. Graphical abstract.