Development of a DC-Biased AC-Stimulated Microfluidic Device for the Electrokinetic Separation of Bacterial and Yeast Cells.

Electrokinetic (EK) microsystems, which are capable of performing separations without the need for labeling analytes, are a rapidly growing area in microfluidics. The present work demonstrated three distinct binary microbial separations, computationally modeled and experimentally performed, in an insulator-based EK (iEK) system stimulated by DC-biased AC potentials. The separations had an increasing order of difficulty. First, a separation between cells of two distinct domains (Escherichia coli and Saccharomyces cerevisiae) was demonstrated. The second separation was for cells from the same domain but different species (Bacillus subtilis and Bacillus cereus). The last separation included cells from two closely related microbial strains of the same domain and the same species (two distinct S. cerevisiae strains). For each separation, a novel computational model, employing a continuous spatial and temporal function for predicting the particle velocity, was used to predict the retention time (tR,p) of each cell type, which aided the experimentation. All three cases resulted in separation resolution values Rs>1.5, indicating complete separation between the two cell species, with good reproducibility between the experimental repetitions (deviations < 6%) and good agreement (deviations < 18%) between the predicted tR,p and experimental (tR,e) retention time values. This study demonstrated the potential of DC-biased AC iEK systems for performing challenging microbial separations.

Manipulating the insulating post arrangement in DC-biased AC-iEK devices to improve microparticle separations.

There is a growing interest in the advancement of microscale electrokinetic (EK) systems for biomedical and clinical applications, as these systems offer attractive characteristics such as portability, robustness, low sample requirements and short response time. The present work is focused on manipulating the characteristics of the insulating post arrangement in insulator-based EK (iEK) systems for separating a binary mixture of spherical microparticles with same diameter (5.1 µm), same shape, made from the same substrate material and only differing in their zeta potential by ∼14 mV. This study presents a combination of mathematical modeling and experimental separations performed by applying a low-frequency alternating current (AC) voltage in iEK systems with 12 distinct post arrangements. These iEK devices were used to systematically study the effect of three spatial characteristics of the insulating post array on particle separations: the horizontal separation and the vertical separation between posts, and introducing an offset to the posts arrangement. Through normalization of the spatial separation between the insulating posts with respect to particle diameter, guidelines to improve separation resolution for different particle mixtures possessing similar characteristics were successfully identified. The results indicated that by carefully designing the spatial arrangement of the post array, separation resolution values in the range of 1.4-2.8 can be obtained, illustrating the importance and effect of the arrangement of insulating posts on improving particle separations. This study demonstrates that iEK devices, with effectively designed spatial arrangement of the insulating post arrays, have the capabilities to perform discriminatory separations of microparticles of similar characteristics.

Assessing the Discriminatory Capabilities of iEK Devices under DC and DC-Biased AC Stimulation Potentials.

There is a rising need for rapid and reliable analytical methods for separating microorganisms in clinical and biomedical applications. Microscale-insulator-based electrokinetic (iEK) systems have proven to be robust platforms for assessing a wide variety of microorganisms. Traditionally, iEK systems are usually stimulated with direct-current (DC) potentials. This work presents a comparison between using DC potentials and using DC-biased alternating-current (AC) potentials in iEK systems for the separation of microorganisms. The present study, which includes mathematical modeling and experimentation, compares the separation of bacterial and yeast cells in two distinct modes by using DC and DC-biased AC potentials. The quality of both separations, assessed in terms of separation resolution (Rs), showed a complete separation (Rs = 1.51) with the application of a DC-biased low-frequency AC signal but an incomplete separation (Rs = 0.55) with the application of an RMS-equivalent DC signal. Good reproducibility between experimental repetitions (<10%) was obtained, and good agreement (~18% deviation) was observed between modeling and experimental retention times. The present study demonstrates the potential of extending the limits of iEK systems by employing DC-biased AC potentials to perform discriminatory separations of microorganisms that are difficult to separate with the application of DC potentials.

Integrated Microfluidic-Electromagnetic System to Probe Single-Cell Magnetotaxis in Microconfinement.

Magnetotactic bacteria have great potential for use in biomedical and environmental applications due to the ability to direct their navigation with a magnetic field. Applying and accurately controlling a magnetic field within a microscopic region during bacterial magnetotaxis studies at the single-cell level is challenging due to bulky microscope components and the inherent curvilinear field lines produced by commonly used bar magnets. In this paper, a system that integrates microfluidics and electromagnetic coils is presented for generating a linear magnetic field within a microenvironment compatible with microfluidics, enabling magnetotaxis analysis of groups or single microorganisms on-chip. The platform, designed and optimised via finite element analysis, is integrated into an inverted fluorescent microscope, enabling visualisation of bacteria at the single-cell level in microfluidic devices. The electromagnetic coils produce a linear magnetic field throughout a central volume where the microfluidic device containing the magnetotactic bacteria is located. The magnetic field, at this central position, can be accurately controlled from 1 to 10 mT, which is suitable for directing the navigation of magnetotactic bacteria. Potential heating of the microfluidic device from the operating coils was evaluated up to 2.5 A, corresponding to a magnetic field of 7.8 mT, for 10 min. The maximum measured heating was 8.4 °C, which enables analysis without altering the magnetotaxis behaviour or the average swimming speed of the bacteria. Altogether, this work provides a design, characterisation and experimental test of an integrated platform that enables the study of individual bacteria confined in microfluidics, under linear and predictable magnetic fields that can be easily and accurately applied and controlled.

Hybrid plasmonic metasurface as enhanced Raman hot-spots for pesticide detection at ultralow concentrations.

A surface-enhanced Raman scattering (SERS) active metasurface composed of metallic nanohole arrays and metallic nanoparticles is developed. The metasurface can operate in aqueous environments, achieves an enhancement factor of 1.83 × 109 for Rhodamine 6G, and enables the detection of malachite green at a concentation of 0.46 ppb.

Fine-Tuning the Characteristic of the Applied Potential To Improve AC-iEK Separations of Microparticles.

There is an immediate need for the development of rapid and reliable methods for microparticle and cell assessments, and electrokinetic (EK) phenomena can be exploited to meet that need in a low cost and label-free fashion. The present study combines modeling and experimentation to separate a binary mixture of microparticles of the same size (5.1 µm), shape (spherical), and substrate material (polystyrene), but with a difference in particle zeta potentials of only ∼14 mV, by applying direct current (DC)-biased low-frequency alternating current (AC) voltages in an insulator-based-EK (iEK) system. Four distinct separations were carried out to systematically study the effect of fine-tuning each of the three main characteristics of the applied voltage: frequency, amplitude, and DC bias. The results indicate that fine-tuning each parameter improved the separation from an initial separation resolution Rs = 0.5 to a final resolution Rs = 3.1 of the fully fine-tuned separation. The separation method exhibited fair reproducibility in retention time with variations ranging from 6 to 26% between experimental repetitions. The present study demonstrates the potential to extend the limits of iEK systems coupled with carefully fine-tuned DC-biased low-frequency AC voltages to perform discriminatory micron-sized particle separations.

Biomimetic macroscopic hierarchical moire gratings.

Large-scale hierarchical macroscopic moire gratings resembling the surface structure of Peruvian lily flower petals are fabricated on azobenzene molecular glass thin films using a Lloyd's mirror interferometer. It is shown that nanostructured linear and crossed moire gratings can be made with pitch values reaching a few millimeters. Also, using atomic force microscopy, scanning electron microscopy, optical microscopy, and surface profilometry techniques, it is shown that the obtained moire gratings have two-fold or three-fold hierarchical structures fabricated using a simple all optical technique.

FDTD Analysis of Hotspot-Enabling Hybrid Nanohole-Nanoparticle Structures for SERS Detection.

Metallic nanoparticles (MNPs) and metallic nanostructures are both commonly used, independently, as SERS substrates due to their enhanced plasmonic activity. In this work, we introduce and investigate a hybrid nanostructure with strong SERS activity that benefits from the collective plasmonic response of the combination of MNPs and flow-through nanohole arrays (NHAs). The electric field distribution and electromagnetic enhancement factor of hybrid structures composed of silver NPs on both silver and gold NHAs are investigated via finite-difference time-domain (FDTD) analyses. This computational approach is used to find optimal spatial configurations of the nanoparticle positions relative to the nanoapertures and investigate the difference between Ag-NP-on-Ag-NHAs and Ag-NP-on-Au-NHAs hybrid structures. A maximum GSERS value of 6.8 × 109 is achieved with the all-silver structure when the NP is located 0.5 nm away from the rim of the NHA, while the maximum of 4.7 × 1010 is obtained when the nanoparticle is in full contact with the NHA for the gold-silver hybrid structure. These results demonstrate that the hybrid nanostructures enable hotspot formation with strong SERS activity and plasmonic enhancement compatible with SERS-based sensing applications.

Cicada Wing Inspired Template-Stripped SERS Active 3D Metallic Nanostructures for the Detection of Toxic Substances.

This article introduces a bioinspired, cicada wing-like surface-enhanced Raman scattering (SERS) substrate based on template-stripped crossed surface relief grating (TS-CSRG). The substrate is polarization-independent, has tunable nanofeatures and can be fabricated in a cleanroom-free environment via holographic exposure followed by template-stripping using a UV-curable resin. The bioinspired nanostructures in the substrate are strategically designed to minimize the reflection of light for wavelengths shorter than their periodicity, promoting enhanced plasmonic regions for the Raman excitation wavelength at 632.8 nm over a large area. The grating pitch that enables an effective SERS signal is studied using Rhodamine 6G, with enhancement factors of the order of 1 × 104. Water contact angle measurements reveal that the TS-CSRGs are equally hydrophobic to cicada wings, providing them with potential self-cleaning and bactericidal properties. Finite-difference time-domain simulations are used to validate the nanofabrication parameters and to further confirm the polarization-independent electromagnetic field enhancement of the nanostructures. As a real-world application, label-free detection of melamine up to 1 ppm, the maximum concentration of the contaminant in food permitted by the World Health Organization, is demonstrated. The new bioinspired functional TS-CSRG SERS substrate holds great potential as a large-area, label-free SERS-active substrate for medical and biochemical sensing applications.

Electrokinetically-Driven Assembly of Gold Colloids into Nanostructures for Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) enables the highly sensitive detection of (bio)chemical analytes in fluid samples; however, its application requires nanostructured gold/silver substrates, which presents a significant technical challenge. Here, we develop and apply a novel method for producing gold nanostructures for SERS application via the alternating current (AC) electrokinetic assembly of gold nanoparticles into two intricate and frequency-dependent structures: (1) nanowires, and (2) branched "nanotrees", that create extended sensing surfaces. We find that the growth of these nanostructures depends strongly on the parameters of the applied AC electric field (frequency and voltage) and ionic composition, specifically the electrical conductivity of the fluid. We demonstrate the sensing capabilities of these gold nanostructures via the chemical detection of rhodamine 6G, a Raman dye, and thiram, a toxic pesticide. Finally, we demonstrate how these SERS-active nanostructures can also be used as a concentration amplification device that can electrokinetically attract and specifically capture an analyte (here, streptavidin) onto the detection site.

Structural Stability of Optofluidic Nanostructures in Flow-Through Operation.

Optofluidic sensors based on periodic arrays of subwavelength apertures that support surface plasmon resonance can be employed as both optical sensors and nanofluidic structures. In flow-through operation, the nanoapertures experience pressure differences across the substrate in which they are fabricated, which imposes the risk for structural failure. This work presents an investigation of the deflection and structural stability of nanohole array-based optofluidic sensors operating in flow-through mode. The analysis was approached using experiments, simulations via finite element method, and established theoretical models. The results depict that certain areas of the sensor deflect under pressure, with some regions suffering high mechanical stress. The offset in the deflection values between theoretical models and actual experimental values is overturned when only the effective area of the substrate, of 450 µm, is considered. Experimental, theoretical, and simulation results suggest that the periodic nanostructures can safely operate under trans-membrane pressures of up to 20 psi, which induce deflections of up to ~20 µm.

Rapid label-free detection of intact pathogenic bacteria in situ via surface plasmon resonance imaging enabled by crossed surface relief gratings.

The unique plasmonic energy exchange occurring within metallic crossed surface relief gratings (CSRGs) has recently motivated their use as biosensors. However, CSRG-based biosensing has been limited to spectroscopic techniques, failing to harness their potential for integration with ubiquitous portable electronics. Here, we introduce biosensing via surface plasmon resonance imaging (SPRi) enabled by CSRGs. The SPRi platform is fully integrated including optics and electronics, has bulk sensitivity of 613 Pixel Intensity Unit (PIU)/Refractive Index Unit (RIU), a resolution of 10-6 RIU and a signal-to-noise ratio of ∼33 dB. Finite-Difference Time-Domain (FDTD) simulations confirm that CSRG-enabled SPRi is supported by an electric field intensity enhancement of ∼30 times, due to plasmon resonance at the metal-dielectric interface. In the context of real-world biosensing applications, we demonstrate the rapid (<35 min) and label-free detection of uropathogenic E. coli (UPEC) in PBS and human urine samples for concentrations ranging from 103 to 109 CFU mL-1. The detection limit of the platform is ∼100 CFU mL-1, three orders of magnitude lower than the clinical detection limit for diagnosis of urinary tract infection. This work presents a new avenue for CSRGs as SPRi-based biosensing platforms and their great potential for integration with portable electronics for applications requiring in situ detection.

Facile actuation of aqueous droplets on a superhydrophobic surface using magnetotactic bacteria for digital microfluidic applications.

Magnetic actuation provides a low-cost, simple method for droplet manipulation on a digital microfluidic platform. The impetus to move the droplets on a low friction surface can come from internal superparamagnetic particles or paramagnetic salts. Recently, the use of microbes for bio-actuation has been established, where the thrust produced by the microbes can be exploited to exert the force required for droplet movement. This study presents biologically-driven magnetic actuation of droplets on a superhydrophobic surface using magnetotactic bacteria (MTB). MTB-droplets were impelled along various trajectories such as rectangular and figure-of-eight-shaped paths. Droplets were reproducibly actuated with speeds up of to 30 mm s-1. We demonstrated the ability to sequentially merge and mix multiple droplets by merging a 10 µL MTB droplet with two 4 µL colored droplets. The reorientation of MTB in the droplet enhanced mixing rate of the merged fluids by ∼40% compared with the control experiment where no actuation was used. Biologically-driven magnetic actuation was compared with actuation by superparamagnetic particles and paramagnetic salts, in terms of controllability and speed. MTB droplet was moved with the same average speed as other two methods and showed higher response time as the magnet acceleration increased. Lastly, MTB were used to perform a phosphatase assay using endogenous enzyme. The relative absorbance at 405 nm, indicating the production of the yellow product, increased over time and levels off after 75 min.

Wavelength-selective plasmonic sensor based on chirped-pitch crossed surface relief gratings.

Two-dimensional chirped-pitch crossed surface relief gratings (CP-CSRGs) were fabricated on azobenzene-functionalized thin films using a simple two-step procedure. The resulting gratings had a constant pitch in one direction and a varying (chirped) pitch in the orthogonal direction. They were coated with silver and tested for their ability to change the polarization of surface plasmon resonance (SPR) signals, when placed between crossed polarizers. It was observed that several different bandwidths of SPR wavelengths are excitable using a single device, making CP-CSRGs suitable as next generation SPR-based sensors. The SPR wavelengths shifted as much as 10.5 nm/mm along the chirped grating, and a maximum sensitivity of 778.6 nm/RIU was obtained when detecting the refractive index change of various concentrations of aqueous sucrose solutions.

Selective Uropathogenic E. coli Detection Using Crossed Surface-Relief Gratings.

Urinary tract infections (UTIs) are one of the major burdens on public healthcare worldwide. One of the primary causes of UTIs is the invasion of the urinary tract by uropathogenic Escherichia coli (UPEC). Improper treatment of bacterial infections like UTIs with broad-spectrum antibiotics has contributed to the rise of antimicrobial resistance, necessitating the development of an inexpensive, rapid and accurate detection of UPEC. Here, we present real-time, selective and label-free detection of UPEC using crossed surface-relief gratings (CSRGs) as nanometallic sensors incorporated into an optical sensing platform. CSRGs enable real-time sensing due to their unique surface plasmon resonance (SPR)-based light energy exchange, resulting in detection of a very-narrow-bandwidth SPR signal after the elimination of residual incident light. The platform's sensing ability is experimentally demonstrated by the detection of bulk refractive index (RI) changes, with a bulk sensitivity of 382.2 nm/RIU and a resolution in the order of 10-6 RIU. We also demonstrate, for the first time, CSRG-based real-time selective capture and detection of UPEC in phosphate-buffered saline (PBS) solution, in clinically relevant concentrations, as opposed to other UTI-causing Gram-negative bacteria. The platform's detection limit is calculated to be 105 CFU/mL (concentration on par with the clinical threshold for UTI diagnosis), with a dynamic range spanning four orders of magnitude. This work paves the way for the development of inexpensive point-of-care diagnosis devices focusing on effective treatment of UTIs, which are a burden on public healthcare due to the rise in the number of cases and their recurrences in the recent past.

Direct Detection of Toxic Contaminants in Minimally Processed Food Products Using Dendritic Surface-Enhanced Raman Scattering Substrates.

We present a method for the surface-enhanced Raman scattering (SERS)-based detection of toxic contaminants in minimally processed liquid food products, through the use of a dendritic silver nanostructure, produced through electrokinetic assembly of nanoparticles from solution. The dendritic nanostructure is produced on the surface of a microelectrode chip, connected to an AC field with an imposed DC bias. We apply this chip for the detection of thiram, a toxic fruit pesticide, in apple juice, to a limit of detection of 115 ppb, with no sample preprocessing. We also apply the chip for the detection of melamine, a toxic contaminant/food additive, to a limit of detection of 1.5 ppm in milk and 105 ppb in infant formula. All the reported limits of detection are below the recommended safe limits in food products, rendering this technique useful as a screening method to identify liquid food with hazardous amounts of toxic contaminants.

Erratum: Publisher's Note: "Migration of magnetotactic bacteria in porous media" [Biomicrofluidics 12, 011101 (2018)].

[This corrects the article DOI: 10.1063/1.5024508.].

Migration of magnetotactic bacteria in porous media.

Magnetotactic bacteria (MTB) migrate in complex porous sediments where fluid flow is ubiquitous. Here, we demonstrate that magnetotaxis enables MTB to migrate effectively through porous micromodels. Directed MTB can circumvent curved obstacles by traveling along the boundaries and pass flat obstacles by repeatedly switching between forward and backward runs. Magnetotaxis enables directed motion of MTB through heterogeneous porous media, overcoming tortuous flow fields with local velocities as high as 250 µm s-1. Our findings bring new insights into the migration behaviour of MTB in their natural habitats and their potential in vivo applications as microbiorobots.

Cost-effective flow-through nanohole array-based biosensing platform for the label-free detection of uropathogenic E. coli in real time.

Rapid, inexpensive and sensitive detection of uropathogenic Escherichia coli (UPEC), a common cause of ascending urinary tract infections (UTIs) including cystitis and pyelonephritis, is critical given the increasing number of cases and its recurrence worldwide. In this paper, we present a label-free nanoplasmonic sensing platform, built with off-the-shelf optical and electronic components, which can detect intact UPEC at concentrations lower than the physiological limit for UTI diagnosis, in real time. The sensing platform consists of a red LED light source, lens assembly, CMOS detector, Raspberry Pi interface in conjugation with a metallic flow-through nanohole array-based sensor. Detection is achieved exploiting nanoplasmonic phenomena from the nanohole arrays through surface plasmon resonance imaging (SPRi) technique. The platform has a bulk sensitivity of 212 pixel intensity unit (PIU)/refractive index unit (RIU), and a resolution in the order of 10-6 RIU. We demonstrate capture and detection of UPEC with a detection limit of ~100 CFU/ml - a concentration well below the threshold limit for UTI diagnosis in clinical samples. We also demonstrate detection of UPEC in spiked human urine samples for two different concentrations of bacteria. This work is particularly relevant for point-of-care applications, especially for regions around the world where accessibility to medical facilities is heavily dependent upon economy, and availability.

Magnetotaxis Enables Magnetotactic Bacteria to Navigate in Flow.

Magnetotactic bacteria (MTB) play an important role in Earth's biogeochemical cycles by transporting minerals in aquatic ecosystems, and have shown promise for controlled transport of microscale objects in flow conditions. However, how MTB traverse complex flow environments is not clear. Here, using microfluidics and high-speed imaging, it is revealed that magnetotaxis enables directed motion of Magnetospirillum magneticum over long distances in flow velocities ranging from 2 to 1260 µm s-1 , corresponding to shear rates ranging from 0.2 to 142 s-1 -a range relevant to both aquatic environments and biomedical applications. The ability of MTB to overcome a current is influenced by the flow, the magnetic field, and their relative orientation. MTB can overcome 2.3-fold higher flow velocities when directed to swim perpendicular to the flow as compared to upstream, as the latter orientation induces higher drag. The results indicate a threshold drag of 9.5 pN, corresponding to a flow velocity of 550 µm s-1 , where magnetotaxis enables MTB to overcome counterdirectional flow. These findings bring new insights into the interactions of MTB with complex flow environments relevant to aquatic ecosystems, while suggesting opportunities for in vivo applications of MTB in microbiorobotics and targeted drug delivery.