Krishna
Kant
a,
Jeongha
Yoo
b,
Steven
Amos
b,
Mason
Erkelens
b,
Craig
Priest
c,
Joe G.
Shapter
a and
Dusan
Losic
*b
aSchool of Chemical and Physical Sciences, Flinders University, Bedford Park, Adelaide 5042, Australia
bSchool of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australia. E-mail: Dusan.losic@adelaide.edu.au; Tel: +61 8 8013 4648
cIan Wark Research Institute, University of South Australia, Mawson Lakes, Adelaide 5095, Australia
First published on 4th March 2015
This paper presents a microfluidic device with a nano-channel prepared by focused ion beam (FIB) milling for microbial cell lysis and nucleic acid extraction. The device prepared in a quartz slide combines two microchannels connected with one nanochannel in the middle with dimensions of 100 nm (width) and 250 nm (depth) and 1 μm (length). The capturing of a single or a few cells and their lysis for extraction of DNA in nanochannel were demonstrated. The purity and quantity of the extracted genetic material was tested using a UV spectrometer. The results revealed that the extraction methodology was successful and may be well-suited to integration in analytical lab-on-a-chip devices.
Coupled with advanced analytical techniques, they offer potential in applications from point of care to single cell analysis,1,2 and other examples where resources are limited.3,4 It was shown that incorporation of biological membranes and synthetic nanochannels in a device can create new opportunities for advanced analytical applications5,6 due to the increased surface area and reduced use of chemical reagents.7,8 Nanofluidic devices may also manipulate cell entities such as proteins, nucleic acids and cell organelles providing ultra-high sensitive analysis using ion source and electrochemical detection.9 Single-cell analysis can be achieved by integration of several biochemical steps into a micro total analysis system (μTAS) on a single chip.10,11 However, it is difficult to handle and manipulate single cells in microsystems, since very small volumes are involved in the analysis.12 The genome of any simple organism may contain thousands of base pairs and it is even more complex in higher species.13,14 Consequently, an integrated nanofluidic chip is a required approach to handling these tiny volumes of complex cell material without damage or loss. Ideally, all steps from cell lysis to analysis of the molecular cell contents, including DNA analysis, are needed on a single integrated chip.15–17 Furthermore, integration of several devices for parallel analysis for different molecules is an attractive prospect.
A number of sophisticated fabrication tools have been used to fabricate nanochannels in recent years.18 E-beam lithography followed by etching has been used to prepare nanochannels (and nanostructures within nanochannels19) with certain limitations of length and depth (depends on reactive ion etching (RIE) techniques). Nanoimprint lithography is capable of producing nanofluidic channels with dimensions of 10 nm in width.11,13,20,21
In this study, we first introduce the potential of cell lysis and extraction of nucleic acids in the nanofluidic device by trapping, lysing, and extracting microbial cells before eluting the genetic material. Moreover, we describe a suitable technique for the fabrication of nanochannels using focused ion beam (FIB) milling. The prepared devices offer an integrated micro- and nanofluidic system, with features that enable several functions performed during chemical analysis (sample preparation, fluid handling and injection, separation, and detection) and new potential for the development of advanced analytical chips.22,23
The schematic of the nanofluidic device is presented in Fig. 1. This scheme shows the flow of algae cells used as a model into the microchannels and diffusion of NaCl through the nanochannel for cell lysis. The collected raw material from cell lysis is analyzed using UV-vis spectrophotometer.
The nanochannel was patterned by FIB milling using a Helios NanoLab 600 Dual Beam instrument (FEI Company) as schematically presented in Fig. 2(ii)–(iv). After photolithography the chip was sputtered with a 40 nm thick Cr layer to have good controls such as preventing from milling progressively deeper and protecting redeposition of material and structural deformations over the ion beam milling and imaging of channel.20 The ion beam energy for milling nanostructure is typically between 10 and 50 keV, with beam currents varying between 1 pA and 10 nA.24 100 nm width and 250 nm depth nanochannel was milled into a quartz substrate through a 40 nm thick Cr film using an ion beam probe with a beam current of 0.17 nA for 20 keV Ga+ ions at normal incidence on milling pattern of 100 nm width and 500 nm depth. Before milling the nanochannel to connect the two microchannels an extension in Fig. 2(iii) of 10 μm was milled to control the distance between the two big microchannels and provide a flat and smooth surface for final milling of nanochannel. The milled nanochannel in Fig. 2(iv) has Gaussian-like beam profile in the cross sections. Operating in this mode, the width of the nanochannel obtained for a given film thickness and ion beam current is highly reproducible. It is controlled by the user-defined width of the scan area. After milling, Cr was removed using ceric ammonium nitrate.
The access ports were drilled using abrasive powder blasting. The substrate and cover plate are cleaned using piranha solution. Their surfaces are activated in an oxygen plasma (Harrick Plasma, 18 W, 10 min), and then they are brought into contact, forming a reversible bond. The bonding is made permanent by heating the device to 1100 °C in a vacuum furnace and holding at this temperature for >10 h.
The processes for cell injection, trapping, lysis, and elution in microchip are shown in Fig. 3 in brief. 1 × 105 cells per ml diluted with deionized (DI) water in Fig. 3(i) was injected into the R1 port of the microfluidic channel by using a syringe pump (Chemyx, Fusion-200) with flow rate of 3 μl min−1. Once the cells were flowing inside the microfluidic channel, 100 mM NaCl solution for osmotic stresses was injected and maintained with flow rate of 5 μl min−1 into the L1 port of the microchannel. In Fig. 3(ii), microalgae cells were trapped in the middle-extended part of the microchannel by changing the flow direction such as from R1 to R2 and from R2 to R1. By changing the direction from L1 to R2 for the flow of NaCl solution, it flowed across the nanochannel and reached to the other side of the microchannel where the cells were trapped. As soon as the NaCl reached to the other microchannel through the nanochannel, cell lysis was performed for 30 s and shown in Fig. 3(iii). After lysis, L1–L2 microchannel was washed by sterilized water with 5 μl min−1 flow rate. Then genetic materials of lysed alga cell were eluted by starting flow from R2 to L1 with 2 μl min−1 in Fig. 3(iv) and collected in an Eppendorf tube.
To determine the quantity and quality of the DNA extracted by the chip, a UV-vis spectrophotometer (Nanodrop, Thermo scientific, DE, USA) was used as a validation method. The quantity of DNA was displayed and purities of nucleic acids were monitored by 260 nm/280 nm absorbance ratio, which also gives the quality of the DNA extraction.25
After making the nanochannel on the chip it was cleaned by use of Cr etchant (ceric ammonium nitrate) and bonded under vacuum at 1100 °C. This quartz nanochannel chip was then used in the specially designed plastic holder with the inlet and outlet micro bore tubing (Fig. 4E). To confirm that nanochannel was working well and the chip was bonded perfectly we performed a small test with the flow of dye (red fruit dye) in the nano channel. In one microchannel red dye was flowed rate of 10 μl min−1 using a syringe pump and the other channel was filled with deionized (DI) water at same rate of flow. Once completely filled with the aqueous dye solution and DI water, the dye solution was pumped through the nanochannels and, after some time, the dye was observed at the left microchannel because the dye diffused by the concentration gradient via nanochannel in Fig. 4F. This demonstrated that the nanochannel was unblocked and able to transport liquid between the channels.
C. vulgaris is a spherical microscopic cell with 2–10 μm diameter and has many structural elements similar to plants.26 Algae cells contain chlorophyll, which in are fluorescent under the green fluorescent light, so the algae cells were observed using fluorescence microscopy and shown in Fig. 5A. For trapping, cells were induced and trapped by the contraction/expansion in the microchannel beak by changing the flow direction at R1 and R2 ports. The trapped algae cells were fixed well into the microchannel beak and shown in Fig. 5B (fluorescence) and Fig. 5C (optical). Once we have the cells trapped in the microchannel, the injection of extra alga cells was stopped and the unwanted cells were removed from the microchannel by washing them with sterilized water. The cross flow of NaCl solution from L1 to R2 was started through nanochannel as schematically shown in Fig. 3(iii). The flow rate was maintained with 2 μl min−1 flow rate to keep the trapped cells in their place. An algae cell is large in size (more than 10 μm) and the low flow rate of NaCl solution does not move the cell. The osmotic stress in high concentration of NaCl induced the rupture of alga cell walls in solution following a sudden reduction in osmotic pressure. Lysis of the alga cells was thus triggered and the cell material was released. After the cell lysis is completed, the direction of flow was changed and cell materials along with genetic material (DNA, RNA) tend to flow through nanochannel towards the other side of microchannel. Due to the narrow size of nanochannel, some parts of lysed cell materials were separated on basis of their size in front of nanochannel. Finally the desired cell materials flowed along the microchannel via nanochannel and were collected in vials for further analysis.
After lysis of the cell, the flow of NaCl solution was stopped in left microchannel (L1–L2) and cross flow of sterilized water was started from microchannel R2 to L1. The cell organelles started moving towards microchannel (R1–R2), but due to small dimension of the nanochannel only small size molecules can pass through. In this way we separated the small molecules like DNA, RNA and protein along with some cell debris from the rest of the cell materials. The separated molecules are collected and used for further quantification of the material and after filtration through a 0.2 μm filter as discussed in the following section.
The presence of DNA, RNA and protein in the collected cell material after lysis was quantified by Nano-Drop 1000 as a UV-vis spectrometer. During the measurement, the sample was analysed at 10 mm path length to report information about the available genetic materials.27 The ratio of absorbance at 260 nm and 280 nm was used to determine the composition of the nucleic acid. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA.23 A ratio of ∼1.8 is generally accepted as “pure” for DNA; a ratio of ∼2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case in conventional methods, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. It is important to note that the actual ratio will depend on the composition of the nucleic acid. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm. In our study, otherwise, we absorb at 230 nm. In our study, otherwise, we did not worry about contamination to measure absorbance of DNA and RNA using UV-vis spectrometer because no chemicals except NaCl for lysis were used. After sample extraction from the nanofluidic device, an additional off-chip filtration was applied to purify genetic materials. The results of A260/280 were 1.84 and 1.97 for RNA and 1.72 and 1.8 for dsDNA before and after filtering, respectively. These ratios are accepted as a pure and the measured data were shown in Table 1. All data are averages from three replicate experiments and the concentration data indicate their standard deviations. After filtering, there was no change in the concentration of DNA. However, more than two thirds of the RNA has been lost possibly due to degration. Based on these results, the nanofluidic device shows easy and efficient lysis of cells and extraction of DNA without organic chemicals within short time.
Type | Conc. (ng ml−1) | A 260 | A 280 | A 260/280 |
---|---|---|---|---|
Unfiltered | ||||
RNA | 3.9 ± 0.6 | 0.098 | 0.053 | 1.84 |
DNA | 3.1 ± 0.3 | 0.062 | 0.037 | 1.72 |
Protein | 38.3 ± 6 | — | 0.038 | — |
Filtered | ||||
RNA | 1.2 ± 0.2 | 0.03 | 0.016 | 1.97 |
DNA | 2.8 ± 0.6 | 0.056 | 0.036 | 1.8 |
Protein | 11.5 ± 3 | — | 0.01 | — |
This journal is © The Royal Society of Chemistry 2015 |