RESUMO
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 â¼14 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.
RESUMO
Insulator-based electrokinetically driven microfluidic devices stimulated with direct current (DC) voltages are an attractive solution for particle separation, concentration, or isolation. However, to design successful particle manipulation protocols, it is mandatory to know the mobilities of electroosmosis, and linear and nonlinear electrophoresis of the microchannel/liquid/particle system. Several techniques exist to characterize the mobilities of electroosmosis and linear electrophoresis. However, only one method to characterize the mobility of nonlinear electrophoresis has been thoroughly assessed, which generally requires DC voltages larger than 1000 V and measuring particle velocity in a straight microchannel. Under such conditions, Joule heating, electrolysis, and the DC power source cost become a concern. Also, measuring particle velocity at high voltages is noisy, limiting characterization quality. Here we present a protocol-tested on 2 µm polystyrene particles-for characterizing the mobility of nonlinear electrophoresis of the liquid/particle system using a DC voltage of only 30 V and visual inspection of particle dynamics in a microchannel featuring insulating obstacles. Multiphysics numerical modelling was used to guide microchannel design and to correlate particle location during an experiment with electric field intensity. The method was validated against the conventional characterization protocol, exhibiting excellent agreement while significantly reducing measurement noise and experimental complexity.
RESUMO
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 â¼14 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.
RESUMO
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.
Assuntos
Biomimética , Nanoestruturas , Nanoestruturas/química , Microscopia de Força Atômica , Microscopia Eletrônica de VarreduraRESUMO
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.
Assuntos
Hemípteros , Nanoestruturas , Animais , Interações Hidrofóbicas e Hidrofílicas , Nanoestruturas/toxicidade , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
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 â¼33 dB. Finite-Difference Time-Domain (FDTD) simulations confirm that CSRG-enabled SPRi is supported by an electric field intensity enhancement of â¼30 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 â¼100 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.
Assuntos
Infecções por Escherichia coli/urina , Ressonância de Plasmônio de Superfície/instrumentação , Escherichia coli Uropatogênica/isolamento & purificação , Desenho de Equipamento , Humanos , Limite de Detecção , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Refratometria/instrumentação , Refratometria/métodos , Ressonância de Plasmônio de Superfície/métodos , Propriedades de SuperfícieRESUMO
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.
RESUMO
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.
Assuntos
Campos Magnéticos , Magnetospirillum/fisiologia , Microfluídica/métodos , Proteínas de Bactérias/fisiologia , Sistemas de Liberação de Medicamentos , Escherichia coli/fisiologia , RobóticaRESUMO
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.
Assuntos
Contaminação de Alimentos/análise , Sucos de Frutas e Vegetais/análise , Malus , Leite/química , Análise Espectral Raman/métodos , Tiram/análise , Triazinas/análise , Animais , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Prata/químicaRESUMO
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.
Assuntos
Óptica e Fotônica/métodos , Escherichia coli Uropatogênica/isolamento & purificação , Refratometria , Análise EspectralRESUMO
Magnetotactic bacteria (MTB) are promising candidates for use as biomicrorobots in biomedical applications due to their motility, self-propulsion, and the ability to direct their navigation with an applied magnetic field. When in the body, the MTB may encounter non-Newtonian fluids such as blood plasma or mucus. However, their motility and the effectiveness of directed navigation in non-Newtonian fluids has yet to be studied on a single-cell level. In this work, we investigate motility of Magnetospirillum magneticum AMB-1 in three concentrations of polyacrylamide (PAM) solution, a mucus-mimicking fluid. The swimming speeds increase from 44.0 ± 13.6 µm/s in 0 mg/mL of PAM to 52.73 ± 15.6 µm/s in 1 mg/mL then decreases to 24.51 ± 11.7 µm/s in 2 mg/mL and 21.23 ± 10.5 µm/s in 3 mg/mL. This trend of a speed increase in low polymer concentrations followed by a decrease in speed as the concentration increases past a threshold concentration is consistent with other studies of motile, flagellated bacteria. Past this threshold concentration of PAM, there is a higher percentage of cells with an overall trajectory angle deviating from the angle of the magnetic field lines. There is also less linearity in the trajectories and an increase in reversals of swimming direction. Altogether, we show that MTB can be directed in polymer concentrations mimicking biological mucus, demonstrating the influence of the medium viscosity on the linearity of their trajectories which alters the effective path that could be predefined in Newtonian fluids when transport is achieved by magnetotaxis.
RESUMO
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.
Assuntos
Saccharomyces cerevisiae , Escherichia coli , Dispositivos Lab-On-A-Chip , Bacillus cereus , Técnicas Analíticas Microfluídicas , Separação Celular/métodos , Bacillus subtilisRESUMO
The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM.
Assuntos
Análise de Célula Única/métodos , Animais , Linhagem Celular , Cricetinae , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Microscopia Eletrônica de Transmissão , Organelas/ultraestruturaRESUMO
Nanohole array-based biosensors integrated with a microfluidic concentration gradient generator were used for imaging detection and quantification of ovarian cancer markers. Calibration curves based on controlled concentrations of the analyte were created using a microfluidic stepped diffusive mixing scheme. Quantification of samples with unknown concentration of analyte was achieved by image-intensity comparison with the calibration curves. The biosensors were first used to detect the immobilization of ovarian cancer marker antibodies, and subsequently applied for the quantification of the ovarian cancer marker r-PAX8 (with a limit of detection of about 5 nM and a dynamic range from 0.25 to 9.0 µg.mL(-1)). The proposed biosensor demonstrated the ability of self-generating calibration curves on-chip in an integrated microfluidic platform, representing a further step towards the development of comprehensive lab-on-chip biomedical diagnostics based on nanohole array technology.
Assuntos
Biomarcadores Tumorais/análise , Técnicas Analíticas Microfluídicas/instrumentação , Neoplasias Ovarianas/diagnóstico , Fatores de Transcrição Box Pareados/análise , Ressonância de Plasmônio de Superfície/instrumentação , Anticorpos Imobilizados/química , Desenho de Equipamento , Feminino , Humanos , Limite de Detecção , Fator de Transcrição PAX8RESUMO
The integration of fluidics and optics, as in flow-through nanohole arrays, has enabled increased transport of analytes to sensing surfaces. Limits of detection, however, are fundamentally limited by local analyte concentration. We employ the nanohole array geometry and the conducting nature of the film to actively concentrate analyte within the sensor. We achieve 180-fold enrichment of a dye, and 100-fold enrichment and simultaneous sensing of a protein in less than 1 min. The method presents opportunities for an order of magnitude increase in sensing speed and 2 orders of magnitude improvement in limit of detection.
Assuntos
Análise de Injeção de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Nanoestruturas/análise , Refratometria/instrumentação , Soluções/análise , Ressonância de Plasmônio de Superfície/instrumentação , Transdutores , Desenho de Equipamento , Análise de Falha de Equipamento , Nanoestruturas/química , Soluções/químicaRESUMO
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.
RESUMO
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.
Assuntos
Nanopartículas Metálicas , Praguicidas , Praguicidas/análise , Análise Espectral Raman , ÁguaRESUMO
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.
RESUMO
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.
Assuntos
Nanopartículas Metálicas , Nanoestruturas , Ouro/química , Nanopartículas Metálicas/química , Nanoestruturas/química , Prata/química , Análise Espectral RamanRESUMO
We quantify the efficacy of flow-through nanohole sensing, as compared to the established flow-over format, through scaling analysis and numerical simulation. Nanohole arrays represent a growing niche within surface plasmon resonance-based sensing methods, and employing the nanoholes as nanochannels can enhance transport and analytical response. The additional benefit offered by flow-through operation is, however, a complex function of operating parameters and application-specific binding chemistry. Compared here are flow-over sensors and flow-through nanohole array sensors with equivalent sensing area, where the nanohole array sensing area is taken as the inner-walls of the nanoholes. The footprints of the sensors are similar (e.g., a square 20 µm wide flow-over sensor has an equivalent sensing area as a square 30 µm wide array of 300 nm diameter nanoholes with 450 nm periodicity in a 100 nm thick gold film). Considering transport alone, an analysis here shows that given equivalent sensing area and flow rate the flow-through nanohole format enables greatly increased flux of analytes to the sensing surface (e.g., 40-fold for the case of Q = 10 nL/min). Including both transport and binding kinetics, a computational model, validated by experimental data, provides guidelines for performance as a function of binding time constant, analyte diffusivity, and running parameters. For common binding kinetics and analytes, flow-through nanohole arrays offer â¼10-fold improvement in response time, with a maximum of 20-fold improvement for small biomolecules with rapid kinetics.