2023 AIChE Annual Meeting

(287n) Particle-Based Biomolecule Detection Systems for Advancements in Assay Workflows and Signal Transduction

Author

Research Interests: Biosensing, particle engineering, medical device design, 3D printing, polymer chemistry, surface chemistry, acoustics, electrokinetics

Oral Presentations:

  1. Electrophoresis of Biospecific Microparticles for Label-Free Biomarker Detection
  2. Sensing and Sound: Biospecific, Acoustic-Responsive Particles for Multiplexed Biomarker Detection

Introduction: Detection of biomarkers, including proteins, small molecules, and other physiologically relevant molecules, is essential for patient diagnosis and disease management. Approaches to detect and quantify biomarkers incorporate three main elements: a target biomarker being detected, a recognition element that specifically identifies the target biomarker, and a transducer that converts biomarker recognition into a measurable output signal. Each of these elements, in combination with other factors such as required equipment and necessary processing steps, affect the overall accessibility, speed, and sensitivity of a biosensing assay. In my graduate research, I have engineered multiple novel particle-based biosensing systems to 1) explore new methods of label-free signal transduction to simplify assays and 2) streamline assay workflows by integration with custom-designed acoustofluidic devices to enhance the accessibility and speed of assays, as detailed below. Notably, several of these systems are currently patent-pending.

Advancements in signal transduction: Recently, numerous active and inactive nano- and microparticle-based biomarker detection assays have been demonstrated. Compared to non-particle-based biomarker detection assays, such as enzyme-linked immunosorbent assays (ELISAs), particle-based assays can enhance biomarker detection by improving sample mixing, reducing required sample volumes, and imparting additional assay tunability. Generally, particle-based assays are distinguished by their output signal, with the most commonly demonstrated classes being electrochemical and optical; thus, biomarker concentration is coupled with changes in electrical signal, solution color, or fluorescence. Reliance on such signals often necessitates use of labeling molecules, as measured signals typically do not directly originate from the target biomarkers. Introduction of these additional labeling components inherently complicates assays by introducing new sources of error and requiring extra steps that increase the time to results. Moreover, because these signals often require complex or nonstandard equipment for signal measurement (e.g., ultraviolet−visible spectrophotometers and fluorimeters), systems that generate simplified and easily measured signals are of great interest.

Here, we demonstrate electrokinetic microparticle-based approaches for label-free, simplified biosensing using both induced-charge electrophoresis (ICEP) and dielectrophoresis (DEP). By leveraging the innate changes to the electrical properties of particle surfaces upon specific capture of target biomolecules, we show direct signal transduction that manifests in a change in electrokinetic particle motion, which can be easily measured by optical microscopy without the use of secondary labels. More specifically, we show preparation of induced-charge electrophoretic microsensors (ICEMs) by functionalization of gold-polystyrene Janus particles with biospecific recognition elements on their gold hemispheres. Upon application of a high frequency (i.e., ~10 kHz) alternating current (AC) electric field to ICEMs suspended in custom electrokinetic propulsion chambers, asymmetric electroosmotic flows are established over the particles, leading to ICEM propulsion perpendicular to the applied field. When biomarkers are captured on the gold hemisphere of the ICEMs, the biomarkers cause a change in particle polarizability that leads to a decrease in the asymmetry of the electroosmotic flows, resulting in ICEM speed suppression. We show the functionalization of ICEMs with both biotin and anti-ovalbumin IgG antibodies for the specific capture of two model biomolecules, streptavidin and ovalbumin, respectively. We demonstrate that the capture of biomolecules leads to direct signal transduction through ICEM speed suppression; at 100 nM SA, ICEM speed is reduced ~46%, as measured by analysis of videos captured by optical microscopy. We additionally show that by tuning the number of ICEMs used in assay, SA can be detected at concentrations as low as 0.1 nM. Relative to assays like ELISA, which can require over 3 hours to conduct, the ICEM-based assay requires only ~1 hour to yield results. The presented ICEM-based assay is currently patent pending. We further explore the use of negative dielectrophoretic(nDEP) trapping of biospecific microparticles in interdigitated electrode chambers as a novel label-free biomarker detection approach. Again, the electrical properties of the surfaces of prepared particles change upon capture of biomarkers, leading to changes in the nDEP trapping force on particles at high AC field frequencies (i.e., 1 MHz) and, consequently, changes in the probability distribution of nDEP trapped particles. These two approaches, which leverage innate properties of target biomolecules and their interactions with particle surfaces, form the foundations for new paradigms of rapid, simple, and label-free biomarker detection with optical microscopy.

Advancements in assay workflows: The simultaneous detection of multiple biomarkers in multiplexed assays can enhance confidence in testing results and diagnoses. However, conventional biomolecule detection assays, such as ELISA, are not suitable for simultaneous detection, and multiple individual assays are often required. Moreover, traditional biosensing techniques often also require complex processing steps that introduce potential points of error and make detection of multiple biomarkers a tedious task that involves significant user engagement. In addition to proteins, small molecules (e.g., molecules under 1000 Da), can serve as important additional indicators of disease progression, metabolic function, and toxin exposure, among other physiological states. However, compared to detection of proteins, which is often aided by capture of target biomarkers by antibody recognition elements, detection of small molecules is more challenging; due to the miniscule size and similarities in structure of small molecules, antibodies typically lack the selectivity necessary for rigorous quantitation of small molecules in biofluids. This limitation often prevents detection of small molecules through traditional assays. Thus, there is a need for simplified and rapid multiplexed biosensing assays that can evaluate the presence and concentration of disparate biomarker types.

To address these challenges, we show a novel biospecific particle-based system for the simultaneous purification and detection of multiple proteins and small molecules from biofluids. One enabling innovation of this technology is the use of a new class of fluorescently barcoded, functional negative acoustic contrast particles (fNACPs) that are modified with antifouling polymers terminated in biorecognition motifs for the specific capture of biomarkers. Due to the poly(ethylene glycol) linkers between the particle surfaces and capture antigen, nonspecific adsorption is extremely low at physiological conditions. Moreover, because of the modular functionalization approach we employ, the fNACPs can be tuned to detect a range of target biomarkers. The second enabling innovation is the use of an acoustofluidic separation device to rapidly purify fNACPs and captured biomolecules from biofluids. This system makes use of acoustofluidic trapping channels, which produce a pressure node along the center of the channels and antinodes along the walls of the channels. fNACPs are forced to the antinodes of the standing wave due to their negative contrast, where they are trapped by secondary acoustic radiation forces. We show that the discriminant forces acting on particles and blood cells enables their efficient (over 99%) separation from whole blood in <60 seconds. To capture antibodies, we show the functionalization of fNACPs with antigen recognition elements; after capture, the antibodies are labeled by secondary fluorescent antibodies and analyzed by flow cytometry. Using this system, we demonstrate the detection of anti-OVA antibodies at picomolar levels (35-60 pM) from whole blood, a sensitivity competitive with commercial ELISAs. Moreover, while ELISA can require 3-5 hours and considerable user engagement to yield results, the fNACP-enabled assay requires only ~70 minutes to detect biomarkers, with only ~10 minutes of this time involving user engagement. To capture and detect small molecules, we functionalize fNACPs with a plant hormone receptor-inspired biorecognition element and reporter system. Upon binding small molecules, the protein recognition element forms a recognition element-small molecule-fluorescent protein reporter complex, enabling detection of small molecules. By fluorescently barcoding discrete populations of fNACPs based on biomarker specificity, we show multiplexed detection of three individual biomarkers in a single assay by flow cytometry. Overall, this fNACP-based, acoustofluidics-enabled assay offers a simple and versatile approach for the rapid, sensitive, specific, and simultaneous detection of a range of target biomolecules, including proteins and small molecules. The methodology for fNACP production and functionalization is currently patent-pending.