Electrochemical micro-aptasensors for exosome detection based on hybridization chain reaction amplification.

Exosomes are cell-derived nanovesicles that have recently gained popularity as potential biomarkers in liquid biopsies due to the large amounts of molecular cargo they carry, such as nucleic acids and proteins. However, most existing exosome-based analytical sensing methods struggle to achieve high sensitivity and high selectivity simultaneously. In this work, we present an electrochemical micro-aptasensor for the highly sensitive detection of exosomes by integrating a micropatterned electrochemical aptasensor and a hybridization chain reaction (HCR) signal amplification method. Specifically, exosomes are enriched on CD63 aptamer-functionalized electrodes and then recognized by HCR products with avidin-horseradish peroxidase (HRP) attached using EpCAM aptamers as bridges. Subsequently, the current signal that is generated through the enzyme reaction between the HRP enzyme and 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 directly correlates to the amount of bound HRP on the HCR products and thus to the number of target exosomes. By introducing anti-EpCAM aptamers, micro-aptasensors can detect cancerous exosomes with high specificity. Due to the micropatterned electrodes and HCR dual-amplification strategy, the micro-aptasensors achieve a linear detection response for a wide range of exosome concentrations from 2.5×103 to 1×107 exosomes/mL, with a detection limit of 5×102 exosomes/mL. Moreover, our method successfully detects lung cancer exosomes in serum samples of early-stage and late-stage lung cancer patients, showcasing the great potential for early cancer diagnosis.

Fabrication of tunable, high-molecular-weight polymeric nanoparticles via ultrafast acoustofluidic micromixing.

High-molecular-weight polymeric nanoparticles are critical to increasing the loading efficacy and tuning the release profile of targeted molecules for medical diagnosis, imaging, and therapeutics. Although a number of microfluidic approaches have attained reproducible nanoparticle synthesis, it is still challenging to fabricate nanoparticles from high-molecular-weight polymers in a size and structure-controlled manner. In this work, an acoustofluidic platform is developed to synthesize size-tunable, high-molecular-weight (>45 kDa) poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) nanoparticles without polymer aggregation by exploiting the characteristics of complete and ultrafast mixing. Moreover, the acoustofluidic approach achieves two features that have not been achieved by existing microfluidic approaches: (1) multi-step (≥2) sequential nanoprecipitation in a single device, and (2) synthesis of core-shell structured PLGA-PEG/lipid nanoparticles with high molecular weights. The developed platform expands microfluidic potential in nanomaterial synthesis, where high-molecular-weight polymers, multiple reagents, or sequential nanoprecipitations are needed.

Acoustofluidic centrifuge for nanoparticle enrichment and separation.

Liquid droplets have been studied for decades and have recently experienced renewed attention as a simplified model for numerous fascinating physical phenomena occurring on size scales from the cell nucleus to stellar black holes. Here, we present an acoustofluidic centrifugation technique that leverages an entanglement of acoustic wave actuation and the spin of a fluidic droplet to enable nanoparticle enrichment and separation. By combining acoustic streaming and droplet spinning, rapid (<1 min) nanoparticle concentration and size-based separation are achieved with a resolution sufficient to identify and isolate exosome subpopulations. The underlying physical mechanisms have been characterized both numerically and experimentally, and the ability to process biological samples (including DNA segments and exosome subpopulations) has been successfully demonstrated. Together, this acoustofluidic centrifuge overcomes existing limitations in the manipulation of nanoscale (<100 nm) bioparticles and can be valuable for various applications in the fields of biology, chemistry, engineering, material science, and medicine.

Acoustofluidics-Assisted Fluorescence-SERS Bimodal Biosensors.

Acoustofluidics, the fusion of acoustics and microfluidic techniques, has recently seen increased research attention across multiple disciplines due in part to its capabilities in contactless, biocompatible, and precise manipulation of micro-/nano-objects. Herein, a bimodal signal amplification platform which relies on acoustofluidics-induced enrichment of nanoparticles is introduced. The dual-function biosensor can perform sensitive immunofluorescent or surface-enhanced Raman spectroscopy (SERS) detection. The platform functions by using surface acoustic waves to concentrate nanoparticles at either the center or perimeter of a glass capillary; the concentration location is adjusted simply by varying the input frequency. The immunofluorescence assay is achieved by concentrating fluorescent analytes and functionalized nanoparticles at the center of the microchannel, thereby improving the visibility of the fluorescent output. By modifying the inner wall of the glass capillary with plasmonic Ag nanoparticle-deposited ZnO nanorod arrays and focusing analytes toward the perimeter of the microchannel, SERS sensing using the same device setup is achieved. Nanosized exosomes are used as a proof-of-concept to validate the performance of the acoustofluidic bimodal biosensor. With its sample-enrichment functionality, bimodal sensing, short processing time, and minute sample consumption, the acoustofluidic chip holds great potential for the development of lab-on-a-chip based analysis systems in many real-world applications.

Deterministic droplet coding via acoustofluidics.

Droplet microfluidics has become an indispensable tool for biomedical research and lab-on-a-chip applications owing to its unprecedented throughput, precision, and cost-effectiveness. Although droplets can be generated and screened in a high-throughput manner, the inability to label the inordinate amounts of droplets hinders identifying the individual droplets after generation. Herein, we demonstrate an acoustofluidic platform that enables on-demand, real-time dispensing, and deterministic coding of droplets based on their volumes. By dynamically splitting the aqueous flow using an oil jet triggered by focused traveling surface acoustic waves, a sequence of droplets with deterministic volumes can be continuously dispensed at a throughput of 100 Hz. These sequences encode barcoding information through the combination of various droplet lengths. As a proof-of-concept, we encoded droplet sequences into end-to-end packages (e.g., a series of 50 droplets), which consisted of an address barcode with binary volumetric combinations and a sample package with consistent volumes for hosting analytes. This acoustofluidics-based, deterministic droplet coding technique enables the tagging of droplets with high capacity and high error-tolerance, and can potentially benefit various applications involving single cell phenotyping and multiplexed screening.

Erratum: Acoustofluidic Synthesis of Particulate Nanomaterials.

[This corrects the article DOI: 10.1002/advs.201900913.].

Correction: Cell lysis via acoustically oscillating sharp edges.

Correction for 'Cell lysis via acoustically oscillating sharp edges' by Zeyu Wang et al., Lab Chip, 2019, 19, 4021-4032, DOI: .

Correction: An acoustofluidic device for efficient mixing over a wide range of flow rates.

Correction for 'An acoustofluidic device for efficient mixing over a wide range of flow rates' by Hunter Bachman et al., Lab Chip, 2020, 20, 1238-1248, DOI: .

Correction: On-chip stool liquefaction via acoustofluidics.

Correction for 'On-chip stool liquefaction via acoustofluidics' by Shuaiguo Zhao et al., Lab Chip, 2019, 19, 941-947, DOI: .

Correction: Acoustic tweezers based on circular, slanted-finger interdigital transducers for dynamic manipulation of micro-objects.

Correction for 'Acoustic tweezers based on circular, slanted-finger interdigital transducers for dynamic manipulation of micro-objects' by Putong Kang et al., Lab Chip, 2020, 20, 987-994, DOI: .

Acoustofluidics-Assisted Engineering of Multifunctional Three-Dimensional Zinc Oxide Nanoarrays.

The integration of acoustics and microfluidics (termed acoustofluidics) presents a frontier in the engineering of functional micro-/nanomaterials. Acoustofluidic techniques enable active and precise spatiotemporal control of matter, providing great potential for the design of advanced nanosystems with tunable material properties. In this work, we introduce an acoustofluidic approach for engineering multifunctional three-dimensional nanostructure arrays and demonstrate their potential in enrichment and biosensing applications. In particular, our acoustofluidic device integrates an acoustic transducer with a sharp-edge-based acoustofluidic reactor that enables uniform patterning of zinc oxide (ZnO) nanoarrays with customizable lengths, densities, diameters, and other properties. The resulting ZnO nanoarray-coated glass capillaries can rapidly and efficiently capture and enrich biomolecules with sizes ranging from a few nanometers to several hundred nanometers. In order to enable the detection of these biomolecules, silver (Ag) nanoparticles are deposited onto the ZnO nanoarrays, and the integrated ZnO-Ag capillary device functions as a label-free plasmonic biosensing system for surface-enhanced Raman spectroscopy (SERS) based detection of exosomes, DNA oligonucleotides, and E. coli bacteria. The optical sensing enhancement of ZnO-Ag capillary is further validated through finite-difference time-domain (FDTD) simulations. These findings not only provide insights into the engineering of functional micro/nanomaterials using acoustofluidics but also shed light onto the development of portable microanalytical devices for point-of-care applications.

A disposable acoustofluidic chip for nano/microparticle separation using unidirectional acoustic transducers.

Separation of nano/microparticles based on surface acoustic waves (SAWs) has shown great promise for biological, chemical, and medical applications ranging from sample purification to cancer diagnosis. However, the permanent bonding of a microchannel onto relatively expensive piezoelectric substrates and excitation transducers renders the SAW separation devices non-disposable. This limitation not only requires cumbersome cleaning and increased labor and material costs, but also leads to cross-contamination, preventing their implementation in many biological, chemical, and medical applications. Here, we demonstrate a high-performance, disposable acoustofluidic platform for nano/microparticle separation. Leveraging unidirectional interdigital transducers (IDTs), a hybrid channel design with hard/soft materials, and tilted-angle standing SAWs (taSSAWs), our disposable acoustofluidic devices achieve acoustic radiation forces comparable to those generated by existing permanently bonded, non-disposable devices. Our disposable devices can separate not only microparticles but also nanoparticles. Moreover, they can differentiate bacteria from human red blood cells (RBCs) with a purity of up to 96%. Altogether, we developed a unidirectional IDT-based, disposable acoustofluidic platform for micro/nanoparticle separation that can achieve high separation efficiency, versatility, and biocompatibility.

An acoustofluidic device for efficient mixing over a wide range of flow rates.

Whether reagents and samples need to be combined to achieve a desired reaction, or precise concentrations of solutions need to be mixed and delivered downstream, thorough mixing remains a critical step in many microfluidics-based biological and chemical assays and analyses. To achieve complete mixing of fluids in microfluidic devices, researchers have utilized novel channel designs or active intervention to facilitate mass transport and exchange of fluids. However, many of these solutions have a major limitation: their design inherently limits their operational throughput; that is, different designs work at specific flow rates, whether that be low or high ranges, but have difficulties outside of their tailored design regimes. In this work, we present an acoustofluidic mixer that is capable of achieving efficient, thorough mixing across a broad range of flow rates (20-2000 µL min-1) using a single device. Our mixer combines active acoustofluidic mixing, which is responsible for mixing fluids at lower flow rates, with passive hydrodynamic mixing, which accounts for mixing fluids at higher flow rates. The mechanism, functionality, and performance of our acoustofluidic device are both numerically and experimentally validated. Additionally, the real-world potential of our device is demonstrated by synthesizing polymeric nanoparticles with comparable sizes over a two-order-of-magnitude wide range of flow rates. This device can be valuable in many biochemical, biological, and biomedical applications. For example, using our platform, one may synthesize nanoparticles/nanomaterials at lower flow rates to first identify optimal synthesis conditions without having to waste significant amounts of reagents, and then increase the flow rate to perform high-throughput synthesis using the optimal conditions, all using the same single device and maintaining performance.

Acoustic tweezers based on circular, slanted-finger interdigital transducers for dynamic manipulation of micro-objects.

Acoustic tweezing technologies are gaining significant attention from the scientific communities due to their versatility and biocompatibility. This study presents acoustic tweezers based on circular, slanted-finger interdigital transducers (CSFITs), which can steer the propagation direction of surface acoustic waves (SAWs) by tuning the excitation frequency. The CSFITs based acoustic tweezers enable dynamic and reconfigurable manipulation of micro-objects using multi-tone excitation signals. Compared to traditional interdigital transducers that generate and control SAWs along one axis, the CSFITs allow for simultaneously generating and independently controlling SAWs propagating along multiple axes by changing the frequency composition and the phase information in a multi-tone excitation signal. Moreover, the CSFITs based acoustic tweezers can be used for patterning cells/particles in various distributions and translating them along complex paths. We believe that our design is valuable for cellular-scale biological applications, in which on-chip, contactless, biocompatible handling of bioparticles is needed.

Cell lysis via acoustically oscillating sharp edges.

In this article, we demonstrate an acoustofluidic device for cell lysis using the acoustic streaming effects induced by acoustically oscillating sharp-edged structures. The acoustic streaming locally generates high shear forces that can mechanically rupture cell membranes. With the acoustic-streaming-derived shear forces, our acoustofluidic device can perform cell lysis in a continuous, reagent-free manner, with a lysis efficiency of more than 90% over a range of sample flow rates. We demonstrate that our acoustofluidic lysis device works well on both adherent and non-adherent cells. We also validate it using clinically relevant samples such as red blood cells infected with malarial parasites. Additionally, the unique capability of our acoustofluidic device was demonstrated by performing downstream protein analysis and gene profiling without additional washing steps post-lysis. Our device is simple to fabricate and operate while consuming a relatively low volume of samples. These advantages and other features including the reagent-free nature and controllable lysis efficiency make our platform valuable for many biological and biomedical applications, particularly for the development of point-of-care platforms.

Acoustofluidic Synthesis of Particulate Nanomaterials.

Synthesis of nanoparticles and particulate nanomaterials with tailored properties is a central step toward many applications ranging from energy conversion and imaging/display to biosensing and nanomedicine. While existing microfluidics-based synthesis methods offer precise control over the synthesis process, most of them rely on passive, partial mixing of reagents, which limits their applicability and potentially, adversely alter the properties of synthesized products. Here, an acoustofluidic (i.e., the fusion of acoustic and microfluidics) synthesis platform is reported to synthesize nanoparticles and nanomaterials in a controllable, reproducible manner through acoustic-streaming-based active mixing of reagents. The acoustofluidic strategy allows for the dynamic control of the reaction conditions simply by adjusting the strength of the acoustic streaming. With this platform, the synthesis of versatile nanoparticles/nanomaterials is demonstrated including the synthesis of polymeric nanoparticles, chitosan nanoparticles, organic-inorganic hybrid nanomaterials, metal-organic framework biocomposites, and lipid-DNA complexes. The acoustofluidic synthesis platform, when incorporated with varying flow rates, compositions, or concentrations of reagents, will lend itself unprecedented flexibility in establishing various reaction conditions and thus enable the synthesis of versatile nanoparticles and nanomaterials with prescribed properties.

Open source acoustofluidics.

Over the past several decades, a litany of acoustofluidic devices have been developed which purport to have significant advantages over traditional benchtop analytical tools. These acoustofluidic devices are frequently labeled as "labs-on-chips"; however, many do an insufficient job of limiting their dependence on the lab. Often, acoustofluidic devices still require skilled operators and complex external equipment. In an effort to address these shortcomings, we developed a low-cost, expandable, and multifunctional system for controlling acoustofluidic devices in the audible to low ultrasonic frequency range (31 Hz to 65 kHz). The system was designed around the readily available Arduino prototyping platform because of its user-friendly coding environment and expansive network of open source material; these factors enabled us to create a system capable of generating high voltage oscillatory signals and controlling microscale flows in acoustofluidic devices. Utilizing the established open source system, we achieved a series of acoustofluidic applications involving the manipulation of fluids and biological objects in a portable fashion. In particular, we used our open source acoustofluidic devices to achieve active rotation of cells and microorganisms, and operation of an acoustofluidic mixing device which has previously shown potential for viscous sample preparation, in a portable fashion. Additionally, using low frequency flexural waves and our portable system, we achieved acoustofluidic separation of particles based on size. It is our hope that the open source platform presented here can pave the way for future acoustofluidic devices to be used at the point-of-care, as well as simplify the operation of these devices to enable resource limited users to leverage the benefits of acoustofluidics in their work.

On-chip stool liquefaction via acoustofluidics.

Microfluidic-based portable devices for stool analysis are important for detecting established biomarkers for gastrointestinal disorders and understanding the relationship between gut microbiota imbalances and various health conditions, ranging from digestive disorders to neurodegenerative diseases. However, the challenge of processing stool samples in microfluidic devices hinders the development of a standalone platform. Here, we present the first microfluidic chip that can liquefy stool samples via acoustic streaming. With an acoustic transducer actively generating strong micro-vortex streaming, stool samples and buffers in microchannel can be homogenized at a flow rate up to 30 µL min-1. After homogenization, an array of 100 µm wide micropillars can further purify stool samples by filtering out large debris. A favorable biocompatibility was also demonstrated for our acoustofluidic-based stool liquefaction chip by examining bacteria morphology and viability. Moreover, stool samples with different consistencies were liquefied. Our acoustofluidic chip offers a miniaturized, robust, and biocompatible solution for stool sample preparation in a microfluidic environment and can be potentially integrated with stool analysis units for designing portable stool diagnostics platforms.

Acoustofluidic devices controlled by cell phones.

Acoustofluidic devices have continuously demonstrated their potential to impact medical diagnostics and lab-on-a-chip applications. To bring these technologies to real-world applications, they must be made more accessible to end users. Herein, we report on the effort to provide an easy-to-use and portable system for controlling sharp-edge-based acoustofluidic devices. With the use of a cell phone and a modified Bluetooth® speaker, on-demand and hands-free pumping and mixing are achieved. Additionally, a novel design for a sharp-edge-based acoustofluidic device is proposed that combines both pumping and mixing functions into a single device, thus removing the need for external equipment typically needed to accomplish these two tasks. These applications serve to demonstrate the potential function that acoustofluidic devices can provide in point-of-care platforms. To further this point-of-care goal, we also design a portable microscope that combines with the cell phone and Bluetooth® power supply, providing a completely transportable acoustofluidic testing station. This work serves to bolster the promising position that acoustofluidic devices have within the rapidly changing research and diagnostics fields.