Rapid identification of bacteria utilizing amplified dielectrophoretic force-assisted nanoparticle-induced surface-enhanced Raman spectroscopy
© Cheng et al.; licensee Springer. 2014
Received: 2 May 2014
Accepted: 19 June 2014
Published: 27 June 2014
Dielectrophoresis (DEP) has been widely used to manipulate, separate, and concentrate microscale particles. Unfortunately, DEP force is difficult to be used in regard to the manipulation of nanoscale molecules/particles. For manipulation of 50- to 100-nm particles, the electrical field strength must be higher than 3 × 106 V/m, and with a low applied voltage of 10 Vp-p, the electrode gap needs to be reduced to submicrons. Our research consists of a novel and simple approach, using a several tens micrometers scale electrode (low cost and easy to fabricate) to generate a dielectrophoretic microparticle assembly to form nanogaps with a locally amplified alternating current (AC) electric field gradient, which is used to rapidly trap nanocolloids. The results show that the amplified DEP force could effectively trap 20-nm colloids in the nanogaps between the 5-μm particle aggregates. The concentration factor at the local detection region was shown to be approximately 5 orders of magnitude higher than the bulk solution. This approach was also successfully used in bead-based surface-enhanced Raman spectroscopy (SERS) for the rapid identification of bacteria from diluted blood.
Conventional bacteria identification in the hospital typically requires several days for blood culture, bacteria plate culture, and Enterotube analysis. This extensive detection time could lead to rises in death rates and increased drug resistance. Over the past decade, DNA-based detection assays such as DNA microarrays and DNA hybridization to identify bacteria have become popular[2, 3]. Antibody-based immunoassays such as the enzyme-linked immunosorbent assay (ELISA) and the Western blot have also been used for detection of microorganisms based on the antibody-antigen interaction. Both DNA-based methods require cell lysing, DNA extraction, DNA amplification, hybridization, and reporter labeling, and antibody-based immunoassays require several complicated steps and long, time-consuming professional operations and are costly because they need elaborate fluorescent/enzyme tagging and sophisticated optical instruments to achieve detection and identification of microorganisms within 12 h. Microfluidic technologies have been popularly employed to reduce the reaction time, required cost, and sample/reagent consumption related to DNA/molecule/bacteria detection due to their miniaturization and high surface area to volume ratio[6, 7]. Bead-based assays have the advantage in regard to high collision rate/probability to accelerate DNA-DNA docking and antibody-antigen reactions, and they have been widely used in DNA hybridization and immunoreactions within microfluidic chips[8, 9].
Raman spectroscopy is a direct detection platform without complicated sample preparations used for rapid analysis of chemical and biological components based on the measurement of scattered light from the vibration energy levels of chemical bonds following excitation. Unfortunately, Raman signals obtained from biological samples are usually very weak, especially in the case of dilute samples. The use of metallic nanoparticles (NPs) attached on the surface of cells, which is a well-known surface-enhanced Raman spectroscopy (SERS) technique, can generate a higher intensity and more distinguishable Raman signal[12, 13]. The generation of coffee-ring effect via droplet evaporation is typically used for the purpose of forming NP-bacteria aggregations naturally[14, 15]. Unfortunately, the uniformity of NP-bacteria aggregates is quite random and produces undesirable variations in the spectra. Antibody-conjugated silver NPs (AgNPs) have been used to adsorb on bacteria to produce NP-bacteria aggregation in order to more effectively induce the SERS effect[17, 18]. However, these expensive antibodies result in additional costs, and the complicated operations and hours required for antibody modification processes limit the advantages of this method. Antibody-conjugated NP SERS detection also has been shown to produce an additional molecular signal involved in the measured spectrum. For bacteria detection, the SERS effect could only occur at the hot junction of the roughened substrate and the bacterial surface. It is difficult to get an enhanced signal with a low variation due to the fact that the laser light must be focused on the hot junction. In addition, the impurity involved in detecting targets in real blood samples and the low signal to noise ratio associated with bio-objects limits the advantages of SERS technology.
Alternating current (AC) dielectrophoresis (DEP) is the electric field-induced motion of objects via dielectric polarization under nonuniform electric fields. DEP has been widely used for biotechnology applications in micro/nanoscale environments, and it offers a number of potential advantages over conventional methods for cell/bacteria manipulation, separation, and concentration[20, 21]. DEP is a flexible tool providing an opportunity to manipulate heterogeneous particles simultaneously. Therefore, the NPs and bacteria could be concentrated to form an NP-bacteria aggregate that serves as a detecting slug for enhancing the Raman spectrum of bacteria. Unfortunately, the DEP force is expressed as a cube function with the particle size (FDEP ~ r3); therefore, it is difficult to use DEP force to manipulate nanoscale objects (r < 100 nm), such as proteins, viruses, and NPs[22, 23].
The platform presented in our work uses a novel concept involving a dielectrophoretic microparticle assembly designed to locally amplify an electric field, and thus, NPs can be manipulated to the surface of microparticles/bacteria in order to conduct an SERS analysis of the bacteria. A simple quadruple electrode with a circular metallic shield at the detection area was designed for separation and concentration of bacteria in the diluted blood and online SERS measurement of the concentrated bacteria, respectively. The bacteria and blood cells (BC) could also be separated based on their different DEP behaviors that depend on their dielectric properties under a specific AC electric field frequency. The challenge of previous works for Raman detection of cells/bacteria/viruses could be addressed through a harmonic combination of the DEP selective tapping of the bacteria from a bacteria-BC mixture and the amplified DEP force-assisted NP-bacteria aggregation used for SERS identification of bacteria.
Chip design, fabrication, and operations
The quadruple electrode array (QEA), compared with other configurations such as interdigitated and castellated electrodes, is easily used for detecting samples in different droplets in order to achieve multiple detections. In quadruple electrodes, the target bacteria can be concentrated at one spot using a negative DEP force to improve detection efficiency even if the bacterial concentration is low. A circular metallic shield was also patterned in the middle region between the quadruple electrodes to reduce the fluorescence noise that could be generated by the laser light penetration of the glass substrate. A 200/35-nm Au/Ti layer was deposited on the glass slides (76 mm × 26 mm and 1 mm thick) using an electro-beam evaporator (JST-10 F, JEOL Ltd., Akishima-shi, Japan). A positive photoresist (AZ 5214, MicroChemicals, Ulm, Germany) was spin-coated on the deposited metal layer, and standard photolithography techniques were employed to determine the designed geometries on the metal layer. After photolithography, wet metal etching was used for microelectrode patterning, and the photoresist was then removed using acetone to complete the microelectrode fabrication.
The bacteria/BC/bacteria-BC suspension sample was placed on top of a quadruple electrode in droplet form, and the motion of the cells was observed under an applied AC field. The DEP behaviors were first characterized by varying the AC frequencies from 100 kHz to 1.2 MHz at a fixed voltage of 15 Vp-p to map the DEP properties. The trapping location of bacteria on the electrode edge or in the middle region between the electrodes indicated whether the bacteria exhibited positive or negative DEP at that applied frequency.
Five-micrometer latex particles (Sigma-Aldrich, St. Louis, MO, USA) were used to form the nanopores via a dielectrophoretic microparticle assembly. Fluorescent latex particles (Sigma-Aldrich, St. Louis, MO, USA) with a diameter of 20 nm were used for the purpose of observing the nanoDEP mechanism. Five-micrometer latex particles (without fluorescence) and 20-nm fluorescent particles suspended in deionized water (DI) water at concentrations of 5 × 106 and 1 × 108 particles/ml, respectively, were used for validation of the nanoDEP mechanism of the simple chip. Staphylococcus aureus (BCRC 14957, Gram positive) and Pseudomonas aeruginosa (ATCC 27853, Gram negative) were cultured on tryptic soy agar (TSA) at 35°C. An isotonic solution, a 300-mM sucrose solution with a low conductivity (approximately 2 μS/cm), was used to adjust the conductivity of the experimental buffer solution. To study the separation and detection of the bacteria from the blood cells, a 1× phosphate-buffered saline (PBS) buffer diluted with the 300-mM sucrose solution in a 1:15 ratio was used for the experimental buffer with a final conductivity of 1 mS/cm, owing to the fact that blood cells are highly sensitive to the osmotic pressure of a solution. Bacteria suspended in the isotonic buffer solution with a concentration of 1 × 107 colony-forming units (CFU)/ml and human blood cells were spiked into the prepared bacteria solution in a ratio of 1:400, giving a final blood cell concentration of 107 cells/ml. Silver nanoparticles with a diameter of 40 ± 4 nm (purchased from Sigma-Aldrich, St. Louis, MO, USA) were spiked into the bacteria-BC sample for SERS detection.
For the purpose of driving DEP forces, a multi-output function generator (FLUKE 284, FLUKE Calibration, Everett, WA, USA) with four isolation channels was used to supply an output voltage range of 0.1 to 20 Vp-p with a frequency range of 0 to 16 MHz. The experiment was observed through an inverted microscope (Olympus IX 71, Olympus Corporation, Shinjuku-ku, Japan), and a fluorescent light source was used to excite the fluorescent nanocolloids. The experimental results were recorded in both video and photo formats using a high-speed charge-coupled device (CCD) camera (20 frames/s, Olympus DP 80, Olympus Corporation, Shinjuku-ku, Japan).
Results and discussion
Finite element simulation
Nanocolloid trapping mechanism using the dielectrophoretic microparticle assembly
Optimal conditions and on-chip SERS identification of bacteria
A novel mechanism for dielectrophoretic trapping of nanoscale particles through the use of a microparticle assembly was demonstrated for the purpose of effectively trapping nanocolloids using the amplified positive DEP force. The amplified electric field is shown to be 2 orders higher than the original middle region, and thus, the DEP force at these local regions can be predicted as 4 orders higher. The appropriate design for this trapping mechanism is one in which the gaps of quadruple electrodes are smaller than 50 μm in order to achieve a sufficient electric field strength needed for manipulating nanocolloids using the amplified positive DEP force. This mechanism was also used for SERS identification of bacteria from diluted blood successfully. The bacteria and blood cells were separated employing their different DEP behaviors, and furthermore, the concentrated bacteria produced an amplified positive DEP force for adsorption of AgNPs on the bacteria surface. The enhancement of SERS was at least 5-fold higher at an optimal AgNP concentration of 5 × 10-7 mg/μl when compared with the normal Raman spectrum. These results demonstrate good spectral reproducibility via dielectrophoresis-assisted AgNP-bacteria sorption. This technique could be readily used for the rapid detection of pathogens in human blood after blood culturing for approximately 12 h. Compared to the current method in the hospital, after blood culturing, this simple and rapid platform could accelerate the detection rate from 2 days to a few minutes. In the future, this approach could be widely used for bead-based hybridization and immunoassays.
This work was supported by the National Science Council of Taiwan (NSC 102-2221-E-492 -001 -MY2, NSC 102-2633-E-168-001 and NSC 101-2218-E-492 -002). We thank Prof. Hsien-Chang Chang for providing the simulation assistance in this work. We also thank the National Nano Device Laboratories for supplying the microfabrication equipment.
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