Glass-composite prototyping for flow PCR with in situ DNA analysis
- First Online:
- Cite this article as:
- Pješčić, I., Tranter, C., Hindmarsh, P.L. et al. Biomed Microdevices (2010) 12: 333. doi:10.1007/s10544-009-9389-2
- 219 Views
In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 µl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 µl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (ΦX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.
KeywordsMicrofluidicsContinuous-flow PCRDNA melting analysisXurographyThermal gradientRapid prototyping
Continuous-flow PCR (cf-PCR) is the microfluidic DNA amplification technique that reverts the polymerase chain reaction (PCR) into a steady-state process (Kopp et al. 1998). By maintaining the device at a single thermal and flow profile over time, simpler controls and faster analysis times result (Crews et al. 2008b). In addition, massively serial DNA testing can be achieved with no cross-contamination between successively amplified samples (Schaerli et al. 2008). Two significant limitations to the versatility of cf-PCR have recently been resolved in the literature. #1—Since the number of thermal cycles is designed into the microfluidic structure, it had not been possible to select the number of PCR cycles for a given sample prior to its subsequent analysis. Obeid et al. responded by designing a cf-PCR device with multiple outlets spaced at intervals along the microfluidic channel, thereby allowing for the PCR product to be removed for analysis after 20, 25, 30, 35, or 40 amplification cycles (Obeid et al. 2003). #2—Another challenge associated with cf-PCR, as well as other micro-PCR technologies, involves the integration of analysis subsystems for characterization of the PCR product. Nakayama et al. demonstrated an in situ fluorescence technique to confirm that amplification was occurring during cf-PCR (Nakayama et al. 2006), although product verification was not attempted. In the past, product verification could only be achieved through a post-PCR analysis. Recently, however, Crews et al. showed that the DNA target can be analyzed while it is amplifying, rather than afterwards (Crews et al. 2008c). This was achieved by performing cf-PCR within a stable thermal gradient, during which the florescence of the PCR mixture was imaged. The cyclic melting of the amplifying DNA was characterized, and used to identify the DNA product. This spatial melting analysis is analogous to a time-domain DNA melting analysis (Zhou et al. 2004), and has recently been shown capable of even single nucleotide polymorphism (SNP) genotyping (Crews et al. 2009).
The specific fabrication techniques used for cf-PCR micro-devices greatly determine their expense and complexity. Glass (Crews et al. 2008b; Kopp et al. 1998; Obeid et al. 2003) and glass/silicon (Schneegass et al. 2001) microfluidic devices have been made using conventional micromanufacturing methods. Such devices are chemically robust and posses optimum thermal characteristics for flow PCR. However, even low production volumes of these devices usually require a significant investment of time and financial resources, as well as the support of a dedicated laboratory infrastructure. Cf-PCR devices have also been fabricated from polymers (Becker and Gartner 2008), such as polycarbonate(PC) (Hashimoto et al. 2004), polydimethylsiloxane(PDMS) (Nakayama et al. 2006), and polymethylmethacrylate (PMMA) (Schaerli et al. 2008). Although polymers are more susceptible to flow-induced thermal variations (Crews et al. 2008a; Hashimoto et al. 2004), the transition of cf-PCR microfluidics to these materials has resulted in a reduction in long-term fabrication costs. However, there is typically a significant initial investment to create the original master, mold, or cast required for polymer fabrication. Therefore, when considering iterative device design and development, the conventional glass, silicon, and polymer fabrication techniques are equally impractical.
A recently developed rapid prototyping technique, xurography, is characterized by a low startup cost in addition to a reduction in the long-term investment of time and resources (Bartholomeusz et al. 2005). Greer et al. estimated that Xurographic microfluidic devices can be fabricated for less than $0.40 USD for materials or for less than $7.00 USD for labor and materials (Greer et al. 2007). In addition, the initial investment for the xurography equipment is less than $2000 USD, and no part of the fabrication protocol requires exposure to hazardous chemicals (HF, HNO3, etc.) or cleanroom processing. This technique involves the patterning of double-sided tapes with a precision-guided cutting blade, after which the thin patterned film is sandwiched between blanks of a thicker substrate material (Sundberg et al. 2007). This rapid prototyping technique has been used to fabricate microfluidic devices capable of time-domain DNA melting analysis (Greer et al. 2007; Sundberg et al. 2007) and spatial DNA melting analysis (Crews et al. 2009). While short-term contact with the exposed adhesive layers does not interfere with DNA melting analysis, Erill et al. found that the adhesive in acrylic tape “induces an acute inhibition of PCR” (Erill et al. 2003).
This current article describes the manufacture and use of cf-PCR microdevices fabricated with the xurography technique. Thin films with a silicone adhesive were used to enhance PCR biocompatibility, and glass substrates were used to enhance the thermal performance of the prototype devices.
2 Experimental setup
The experimental system consisted of a microfluidic chip, a syringe and syringe pump (KDS100, KD Scientific, MA, USA), a heating subsystem, and an optical subsystem. A computer was used to store and analyze the images acquired by the optical subsystem. The microfluidic chip had a serpentine channel through which a PCR mixture was pumped. A stable temperature gradient was generated across the chip, such that the flowing fluid would experience the cyclic heating and cooling for PCR to occur. A syringe containing the PCR sample was attached through 1/16” tubing (Upchurch scientific, WA, USA) to the chip inlet. The chip outlet was connected to a waste reservoir. Periodically during the PCR, the fluorescence of the intercalating dye (LC Green Plus, Idaho Technology, UT, USA) in the PCR mixture was imaged. Both the amplification curve and characteristic melting curves of the DNA were obtained from each image.
2.1 Microfluidic device design and fabrication
The thermal heating and cooling cycles were performed as the molecules in the fluid moved between the hottest periphery of the microfluidic channel and its coolest periphery. The product extension by DNA polymerase, which occurred at low to intermediate heating temperatures, was considered the only time-dependent aspect of PCR kinetics (Wittwer and Hermann 1999). While this rate of reaction restricted the maximum heating rate of the PCR mixture, only physical limitations dictated the maximum cooling ramp rate from the denaturing to the annealing temperature. For this reason, wide heating sections (decreasing mixture velocity) and narrow cooling sections (increasing mixture velocity) were characteristic of every tested design. The channel geometries for the iterative design and fabrication were created in Adobe Illustrator (Adobe Systems, CA, USA). At least one microfluidic device of each design was fabricated and tested. Several criteria were used to optimize the design, such as the propensity to form or trap air bubbles, ease of fabrication, the maximum number of channels that would fit on a device, the ramping ratio between heating and cooling sections, and the quality as well as quantity (i.e. spatial redundancy) of fluorescent data.
2.2 Temperature control subsystem
The temperature control subsystem was designed to generate a stable, controllable temperature gradient within the device that would be virtually linear in the vicinity of the microchannel. The uniformity of the thermal gradient would ensure a reliable thermocycling protocol for the PCR as well as a precise DNA melting analysis of the product during its amplification. The thermal subsystem was comprised of a heating platform and a closed-loop temperature controller. A 3D model of the heating platform, along with further assembly and circuitry details about the temperature control subsystem are provided as “Supplementary material”. An infrared (IR) camera (A320, FLIR, OR, USA) with a 320 × 240 pixel array was used during the development of the temperature control subsystem to characterize the spatial temperature distribution across the glass substrate. Prior to using the IR camera, manufacturer’s instructions were followed to calculate the emissivity of the glass device.
2.3 Optical subsystem
The optical subsystem consisted of a diffuse LED light source and a CCD camera, designed to excite and detect the spatial emission of the fluorescent dye that would bind to the DNA as it was amplifying. LC Green Plus absorbs light between 440–470 nm and, when double-stranded DNA is present, strongly emits light between 470–520 nm. Therefore, the LED source (HPLS-Dragon, LightSpeed Technologies, CA, USA) was filtered to a 50 nm wide band between 425–475 nm (HQ450/50x, Chroma, VT, USA).12-bit images of the microfluidic device were acquired with the 0.3 megapixel monochrome camera (LU075, Lumenera, ON, Canada) fitted with a 50 mm macro lens (Canon EF 50 mm f/2.5, Canon, Tokyo, Japan) that was filtered to block out all wavelengths below 485 nm (HQ485LP, Chroma, VT, USA). The field of view for this camera covered approximately ten PCR cycles, with a resolution on the order of 35 µm per pixel. When examination of more cycles was desired, the heating platform and microfluidic device were shifted beneath the stationary optics, so that multiple regions of the chip could be imaged in quick succession. These images were merged together using an automated function in Adobe Photoshop (Adobe Systems, CA, USA), resulting in a single image of the entire channel length.
2.4 Image analysis
A software program was used to obtain DNA amplification and melting data from the fluorescent images. These in-house graphical user interfaces (GUIs) were developed in MATLAB (The MathWorks, MA, USA), based partially on algorithms that have been previously described (Crews et al. 2008c). The program allowed for the user to interactively orient each image and indicate the regions of interest for the analysis.2 Fluorescence was measured as a function of cycle number and of temperature in order to generate an amplification curve and one or more DNA melting curves for each image.
In the amplification curve GUI, it was important to minimize the effect of any spatial variations in the excitation light. Therefore, the background fluorescence in each cycle was subtracted from the measured signal before the amplification curve was obtained. The fluorescence intensity was averaged within a 10 × 10 pixel region of interest within each section to obtain these values. The melting analysis GUI allowed for the user to select any PCR cycle or multiple cycles in which to examine the amplifying DNA. The analysis algorithm took advantage of the spatial redundancy which was inherent in the 2-D fluorescence distribution. In channel sections that were oriented parallel to the 1-D temperature gradient, it was assumed that adjacent pixel lines would exhibit virtually identical fluorescence, differing only by the effects of the optical and electrical noise in the system. By grouping these parallel data lines together for combined analysis, it was hypothesized that the signal-to-noise ratio (SNR) of the data would increase. Therefore, the GUI allowed the user to select the pixel width of the data subset for the spatial DNA melting curve analysis.
2.5 PCR reagents
An identical PCR mixture was used for all of the experiments conducted as part of this work. Unless otherwise noted, all reagents were purchased from Sigma-Aldrich (MO, USA). The mixture contained 108 copies/µl of a viral phage DNA template (ΦX174, New England Biolabs, MA, USA), 0.5 µM of each of the forward and reverse primers (Integrated DNA Technologies, IA, USA), 200 µM of each deoxynucleotide triphosphate (dNTP), 0.04 U/µl of KlenTaq1 polymerase (AB Peptides, MO, USA), 2 mM MgCl2, 2.5 mg/ml BSA, and 1X LCGreen Plus in a 30 mM Tris (pH 8.3) buffer. One of two primer sets3 was used at a time to target either a 110-bp or a 181-bp segment of the template DNA. The concentration of the DNA template used in these tests was approximately 1,000 times higher than is used in clinical applications with human genomic DNA (Crews et al. 2008c). This was done to reduce the number of cycles by ten that would result in a measurable fluorescence signal (10 cycles∼210 increase). Positive and negative controls for each batch of PCR mixture were amplified and analyzed on a commercial system (LS-32, Idaho Technology, UT, USA). The amplification protocol for the LS-32 consisted of a 1 min initial denaturation at 95°C, followed by 30 cycles of 95°C for 1 s, 60°C for 1 s, and 75°C for 3 s. All temperature ramping on the LS-32 was at a rate of 5°C/s. At the conclusion of the PCR, a high resolution melting analysis of each amplified sample was performed serially, by monitoring the fluorescence during a steady ramp of 0.3°C/s from 60°C to 90°C.
Previous work indicated that reactants in the mixture can adhere to the glass walls of the cf-PCR channels, thereby inhibiting initial amplification (Crews et al. 2008c). To reduce this effect in the current system, the microchannel was passivated prior to its first use with a solution identical to the PCR reaction mixture, minus the nucleic acids and Taq polymerase. Forty µl of this passivation mixture was passed through the device at a flow rate of 2 µl/min, and PCR testing was initiated shortly after the microchannel was emptied.
2.6 Microfluidic PCR and analysis
To prepare each device for testing, a microfluidic chip was placed in the heating platform and the heating protocol was initiated. While the gradient was equilibrating, the passivation mixture was passed through the channel and the PCR mixture was loaded into a syringe. At the conclusion of the pre-treatment, the sample was injected into the channel at a constant flow rate of 2 µl/min. The device was periodically imaged while the microchannel was filling. Upon filling, all thermal and flow conditions were maintained. Fluorescence images continued to be taken at intervals, in order to observe any change in the behavior of the system over time. This experiment was repeated multiple times, with multiple chips, and with each of the target-specific primer sets.
Experiments were also conducted to evaluate the thermal stability of the temperature distribution within the glass-polymer-glass microfluidic devices. The melting of the PCR product was used as the metric, such that a shifting or a distortion of the observable fluorescence transition would be indicative of flow-induced temperature drift. Volumetric flow rates up to 4 µl/min were examined. For these tests, a microfluidic device was filled with pre-amplified PCR product of 110-bp in size. When the chip reached thermal equilibrium, the flow rate was set to a value, and 60 s (± 5 s) elapsed before the device was imaged and the flow rate was changed. This was repeated for all of the flow rates of interest.
2.7 Spatial data quality
The effect of spatial redundancy and of camera exposure time on the data quality obtained from the CCD images was investigated by measuring the SNR of the spatial DNA melting curves. These calculations were performed by importing the GUI-generated melting curves into an analysis software (MeltingWizard, University of Utah, UT, USA). A representative amplification of the 110-bp plasmid target was examined for this evaluation. With a full chip, under steady-state flow and thermal conditions, three images of the fluorescence were taken with exposure times of 125 ms, 250 ms, and 1,000 ms. The MATLAB GUI was used to perform spatial melting analyses on each of these images at five arbitrary positions within the 22nd PCR cycle. Line clusters from one to 13 pixels wide were evaluated, and the corresponding SNRs of these curves were examined as a function of exposure time and pixel line width.
3 Results and discussion
3.1 Iterative device design and fabrication
Microfluidic chips were fabricated according to the aforementioned protocol. The final microchannel design, as shown in Fig. 1, contained 23 PCR cycles. During the design development process, device variations were quickly applied and tested. The fabrication of each iterative design was achieved in less than one person-hour, beginning with the initial concept idea and ending with an experiment-ready prototype. The fabrication yield with this technique was approximately 80% when all geometric features were at least 200 µm in size. The occasional unusable prototypes were characterized by wrinkles in the tape leading to leaking, shifting of the serpentine pattern in the tape resulting in channel blockage, and/or leaking around the PDMS ports. Mishandling of the components and inadequate cleaning during fabrication have been identified as the leading causes of these failures.
Figure 1(b) shows the periodic geometry that resulted from the iterative design and testing process. The channel sections where sample cooling would occur were designed to be as narrow as possible. Channel widths below 150 µm, however, proved challenging to fabricate consistently, due to the difficulty associated with manually removing the material on the interior of the cut channel outline. In the final design, the widths for the cooling channels were 200 µm to increase the fabrication yield. In addition, abrupt width changes resulted in more frequent formation of bubbles, and were avoided in the later design iterations. To maximize the extension time for a given volumetric flow rate, gradually increasing widths, up to a maximum of 1.0 mm in the middle of the chip, were selected for the final device design. The thermocycling protocol resulting from this optimized design is shown in Fig. 1(c), which represents the analytical solution derived from the local average fluid velocity. A volumetric flow rate of 2 µl/min was assumed, as well as a linear temperature gradient within the channel from 60°C to 90°C. The total time for each thermal cycle was calculated to be 26 s. Within this cycle, the PCR constituents would be within an acceptable temperature range for polymerase extension (70–80°C) for roughly 9 s, which is more than adequate for full extension of small amplicon. In the optimized design, the two final heating channel sections were a particularly wide 1.5 mm. During the analyses of the fluorescence images, these two channels were the ones most commonly used for the spatial melting analysis, and therefore designed for maximum spatial data redundancy.
Use of the aminosilane coated microscope slides increased the material cost per device by approximately $1.30 USD over the $ 0.40 USD estimated by Greer (Greer et al. 2007). The disposable PDMS interfacing used here adds an additional $0.20 USD material cost per device, although the reusable Nanoport interfaces (∼$20 USD, Upchurch Scientific, WA, USA) suggested by Greer were not included in their estimates. Since the labor involved in mounting the PDMS interfaces versus the Nanoports is similar, the labor costs given by Greer would also apply to this present device manufacture. Therefore, the estimated cost to fabricate the prototype devices described in this article is less than $2 USD for materials, and $10 USD with labor.
3.2 Thermal control and uniformity
By heating a microfluidic device from room temperature with the heating platform and controller, a stable temperature gradient would develop within 5 min, as determined by the IR images of the system. Figure 2(a) shows an IR image after thermal equilibrium was achieved, with a thermal resolution of approximately 400 µm per pixel. In this overhead view, the gradient is oriented vertically, with the cooler edge along the bottom of the chip. The dotted rectangle overlaid on the image in Fig. 2(a) indicates the position within the microfluidic device of the serpentine channel. As is shown in Fig. 2(b), the gradient within this rectangular region encompassed the entire temperature range within which PCR can be achieved. The error bars in the graph show the maximum temperature variation across the rectangular region indicated in Fig. 2(a), with a maximum error of less than 1°C at the lower temperatures, and of approximately 1.4°C at the higher temperatures. With the cross-chip gradient being on the order of 3°C/mm, precise placement of the microfluidic device within the heating platform was critical to reducing thermal variations between experiments. Therefore, the alignment features designed into the heating platform (see “Supplementary material”) proved particularly useful.
3.3 Simultaneous amplification and analysis
The amplification curve, as shown in Fig. 3(b), included one data point for each cycle. Each data point was obtained by examining the intensity of a pair of 10 × 10 pixel squares, one at a vertical position where the product was double-stranded (labeled “signal” in the figure), and the other at a vertical position where the product was single-stranded (labeled “background” in the figure). Since the analysis is of a 12-bit image rather than raw photodiode data, it was not feasible to divide the signal value by the background value. Instead, the difference between these two values (signal minus background) was used as the data point in the amplification curve shown in Fig. 3(b). A first order Savitzky-Golay filter was also used to obtain a best-fit line through the data points. The resulting curve is also shown in the figure. Spatial melting curves, shown in Fig. 3(c), were obtained from cycles 10, 14, 16, 18, and 22 of the image shown in Fig. 3(a). Using the GUI, a 13 pixel wide vertical line of fluorescence intensity was used to produce the DNA melting curves shown. Although a single vertical pixel line could be used to characterize the melting within a channel section, this 13-fold spatial redundancy resulted in a significant increase in the SNR, which approached 200 for higher cycle melts. The curves shown in Fig. 3(c) constitute the raw fluorescence data as described. No additional smoothing was performed.
3.5 Signal-to-noise ratio
It is valuable to note that this inclusion of adjacent pixel lines in the spatial melting analysis—in contrast to averaging in the direction of the gradient—only strengthens the melting signal, and has a clarifying effect, rather than a smudging effect on the data (see Fig. 7(c)). This benefit is a product of the spatial redundancy naturally inherent in such systems, and is independent of the time of experiment.The relatively high SNR obtained with this system is very promising, given the grade of the CCD camera used for this work. By upgrading the optical system with a larger CCD pixel array, or by cooling the CCD chip, it is anticipated that the SNR would increase substantially. Even without these improvements, however, the current version of this system can achieve a SNR with a 1 s exposure time that exceeds the SNR obtained by some commercial systems after an hour long DNA melting analysis (Herrmann et al. 2006).
A low cost microfluidic device for simultaneous DNA amplification and identification was fabricated using a rapid prototyping technique. An iterative design, testing, and re-design process was achieved with minimal time and expense. The microfluidic device was comprised of a patterned polymer film adhered between two un-patterned microscope slides. The final product was a composite structure of approximately 96% glass, thereby maintaining essentially the same thermal and biocompatibility properties of glass. By inducing a steady-state thermal gradient in the substrate through which the PCR mixture was steadily pumped, temperature cycling was performed and efficient, specific amplification was achieved. A 23-cycle PCR was achieved on the device within 15 min, during which the amplifying plasmid target was detected and positively identified. This was done by quantifying the spatial fluorescence distribution produced by an intercalating dye in the mixture. Signal-to-noise ratios on the order of 200 were achieved with an entry-level CCD camera by evaluating areas of fluorescence rather than lines of fluorescence.
With the fabrication technique used in this work, the necessary expense of cf-PCR development and testing is significantly reduced. The virtually unmatched ease of iterative design is coupled with lower cost materials and equipment, while preserving much of the functional advantages of glass microfluidics. The experimental technique presented in this article incorporates the analytical features of real-time PCR into the cf-PCR platform. In addition, the PCR can become the final process of a genetic test, with the DNA being characterized while still within the cf-PCR channels themselves. The in situ melting analysis of the PCR product can be achieved by analyzing data obtained before the reaction is even completed, without interrupting the amplification process. By incorporating such optical analysis, cf-PCR now achieves a significant versatility, which is a welcome companion to its already proven speed and simplicity.
A short video demonstrating the fabrication and attachment of the PDMS ports is located at http://www2.latech.edu/∼ncrews/Presentations.
A recent version of this MATLAB program and an operator’s tutorial is available for download at http://www2.latech.edu/∼ncrews.
110-bp (F—GGTTCGTCAAGGACTGGTTT, R—TTGAACAGCATCGGACTCAG). 181-bp (F – GCTTCCATGACGCAGAAGTT, R – GCGAAAGGTCGCAAAGTAAG)
Authors credit funding by Louisiana Tech University and the Louisiana Space Consortium (LaSPACE). Authors thank Jimmy Cook for machine shop support, Debbie Wood, Dee Tatum, and the rest of the technical staff at the Institute for Micromanufacturing for their support on this work. Special thanks is also given to Dr. Rastko Selmic for his role in the formation of this research team, and Dr. Eric Gilbeau for his mentorship and assistance with the buildup of the research laboratory.