Article pubs.acs.org/Langmuir
Surface-Scribed Transparency-Based Microplates Xin Ye Li,† Brandon Huey-Ping Cheong,† Anthony Somers,‡ Oi Wah Liew,§ and Tuck Wah Ng*,† †
Laboratory for Optics, Acoustics, & Mechanics, Department of Mechanical & Aerospace Engineering, Monash University, Clayton, Victoria 3800, Australia ‡ Institute for Technology Research Innovation (ITRI), Deakin University, 221 Burwood Highway, Burwood, VIC 3125, Australia § Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599 ABSTRACT: Transparency sheets, which are normally associated with use on overhead projectors, offer lowered costs and high amenability for optical diagnostics in microplate instrumentation. An alternative microplate design in which circles are scribed on the surface of the transparency to create the boundaries to hold the drop in place is investigated here. The 3D profile of the scribed regions obtained optically showed strong likelihood of affecting three-phase contact line interactions. During dispensation, the contact angle (≈95°) was larger than the drop advancing state (≈80°) due to a period of nonadhesion, where the contact angle later reduced to the drop advancing state followed by increase in the liquid area coverage on the substrate. It was established that 50 μL was needed to fill the well fully, and the maximum volume retainable before breaching was 190 μL. While the tilt angle needed for displacement reduced significantly from 50 to 95 μL, this was markedly better than nonscribed surfaces, where tilt angles always had to be kept to within 30°. It was found that there was greater ability to fill the well with smaller volumes with dispensation at the center. This was attributed to the growing contact line not meeting the scribed edge in parallel if liquid was dispensed closer to it, wherein pinning reduction in some directions permitted liquid travel along the scribed edge to undergo contact angle hysteresis. Fluorescence measurements conducted showed no performance compromise when using scribed transparency microplates over standard microplates.
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with the use of fluorescence, provide arguably the highest sensitivity measurements with greatest versatility. For this reason they are used extensively in protein formulation and characterization studies and are the method of choice in high throughput screening due to high sensitivity and speed.7−9 Transparency sheets, which are normally associated with use on overhead projectors, have recently been reported for use as microplates.10 This approach offers the features of lowered costs and more importantly high amenability for optical diagnostics which is not feasible when paper or thread media is used. The essence of the method (Figure 1a) is to create an array of holes on a hydrophobic sheet which could then be affixed to the transparency. This disposable unit can then be attached temporarily onto a reusable plexiglass base. The primary purpose of hydrophobic and hydrophilic regions is to keep the analyte within the confines of each well. Nevertheless, it is also desirable to provide the constrained analytes with stability. The ability of liquid drop to remain in a stationary position is dependent on the pinning of its contact line. A contact line is
INTRODUCTION Analytical research and clinical diagnostic screening requires effective fluid handling implements. Standard microplates, which are essentially test tubes in an array on molded plastic plates, are the tools of choice for this, hence their ubiquity. A clear direction forward in microplate instrumentation is for more effective ways to dispense and manage the testing of increasingly smaller liquid volumes.2 Smaller test volumes (i) increase the number of assays that can be conducted per plate thereby increasing throughput and (ii) reduce sample quantity needed per assay which is crucial when the test samples/ reagents are scarce or expensive. In dealing with miniaturized assays, alternative approaches have been developed to handle small liquid volumes, their preparation, and testing without the need for complex or precise machinery.2−4 Yet, there is also impetus to create microplates that are cost-effective enough to be available for use in resource-limited laboratories so that diagnostic outcomes can be achieved in a more timely fashion. Paper and thread, by virtue of their strong wicking capabilities and low cost, are media that have been developed to address this exigency.5,6 Apart from good retention features for small liquid volumes, the ability for effective signal creation and its detection is crucial in microplate instrumentation. Optical approaches, in particular © 2012 American Chemical Society
Received: November 5, 2012 Revised: December 4, 2012 Published: December 7, 2012 849
dx.doi.org/10.1021/la304394s | Langmuir 2013, 29, 849−855
Langmuir
Article
compared with 96-well (Sarstedt Australia Pty Ltd., Product No. 83.9923.974) standard microplates which were circular in shape and had a flat bottom. Milli-Q water was used in all tests related to drop dispensation and stability. For fluorescence quantification, the experimental sample used was enhanced green fluorescent protein (EGFP) carrying a C-terminal polyhistidine tag, isolated from genetically modified Escheria coli and purified by immobilized metal affinity chromatography. After elution of the proteins from the chromatographic matrix, the sample was desalted into sodium phosphate buffer (pH 7.4), checked for purity by SDS-PAGE (sodium dodecyl sulfate−polyacrylamide gel electrophoresis), and quantified using the BCA (bicinchoninic acid) protein assay (Pierce). Sodium phosphate (NaPO4) buffer was used to prepare a series of dilutions for the purified EGFP sample ranging from 65 to 1300 ng/μL. These solutions were delivered using a manual pipet (Biohit mLine Mechanical Pipette, 10−100 μL). Surface Characterization. The scribed transparency was characterized using a 3D optical profilometer (Bruker, Veeco-Wyko) based on a noncontact GT-K1 interferometric system. The instrument was located on a pneumatic vibration-isolation table (Newport) which was fitted with active high attenuation isolation and calibrated using step height standards (Bruker, Veeco). To increase resolution, the green band light source was used for imaging. Analysis was done using the bundled Vision 64 (Ver. 5.10) software. Imaging was done using low and high magnifications in order to derive optimal scanning results. An important feature that we utilized was automatic image stitching which permitted us to combine high-magnification images together to obtain profiles of extended spatial areas. Drop Dispensation Studies. The liquid dispensation studies were conducted on a drop shape analysis system (Kruss, DSA100). The variation of the droplet shape and size with the volume, height, and position of dispensation was investigated. The DSA100 was able to precisely dispense volumes of water with a speed of 300 μL/min and measure the contact angle of the water droplet on the well. The volume analysis was performed for volumes between 20 and 190 μL, in increments of 5 μL, with the needle over the center of the well and at a height of 1.5 mm above the microplate. The evolution of the contact angle for a volume of 190 μL was also measured. For the height analysis, 125 μL of water was dispensed onto a well at various heights between 0.5 and 3.5 mm (in increments of 0.5 mm) above the well. The position analysis investigated the variation of the minimum amount of water to fill a well at seven different points of dispensation across the diameter of the well. The evolution of the contact angle for 30 μL of water was also measured at the different injection sites. Drop Stability Studies. A tilting device was constructed out of an opto-mechanical rotary stage (Newport RSA-1T) with 1° graduations for the stability analysis tests. Specific volumes of water were dispensed onto the microplate using a manual pipet (Eppendorf), and then the tilt table manually turned slowly, in a clockwise and anticlockwise direction, until the fluid displaces over the well. The amounts dispensed ranged from 30 to 95 μL at intervals of 5 μL. Experiments were done on transparencies with and without scribing. Failure of the surface to retain the drop was taken as the situation when the advancing contact line was breached. It has been previously established that the advancing contact line breaches first before the receding contact line.19,20 Fluorescence Quantification. The fluorescence measurements were accomplished using the G:Box Chemi XT4 (SYNGENE) with excitation at a 480 nm wavelength from epi-lighting using blue LEDs. The samples were manually pipetted onto the microplate in duplicates, and a short pass filter (495−600 nm) was used to distinguish the various concentrations of the EGFP samples. The reagent volume used for each well was kept at 60 μL. This was done for both the surfacescribed transparency-based microplate and a standard 96-well microplate. The GeneSnap and GeneTools software were used respectively for the image capture and fluorescent imaging analysis.
Figure 1. The (a) erstwhile hydrophobic sheet with holes cut out and attached to a transparency and (b) the proposed transparency scribed with circles that function as microplates. Both designs (which are disposable) are attached to a plexiglass base (that is reusable).
governed by the ability of the contact angles associated with it to vary over a finite range. It will not advance or recede on a flat surface until the contact angles reach their critical values. If we consider an obstacle present at the contact line, its tilted edge will cause an apparent critical advancing angle θ′a that is larger than the angle without the obstacle θa (Figure 2a,b). The tilted
Figure 2. A droplet on a flat surface has (a) a critical advancing contact angle θa that is increased to (b) θ′a in the presence of an obstacle. Alternatively, it has (c) a critical receding angle θr that is reduced to (d) θ′r in the presence of an obstacle.
edge of the obstacle will, alternatively, cause an apparent critical receding angle θ′r that is smaller than the angle without the obstacle θr (Figure 2c,d). This will then require that θ′r ≤ θ ≤ θ′a
(1)
This inequality was first advanced by Gibbs in which the apparent advancing angle was arguably first demonstrated with pedestals interacting with the liquid body.12 Following that, numerous experiments have been conducted to establish how contact line pinning is affected by surface topologies13−18 and even by bubbles.19,20 The objective of this work, based on the knowledge that the presence of obstacles can strongly affect drop motion, is to investigate an alternative microplate design in which circles are scribed on the surface of the transparency to create the boundaries to hold the drop in place (Figure 1b). The advantages offered by this approach are (i) increased stability in liquid retention and (ii) reduction in cost and time needed to fabricate the transparency microplates. 11
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MATERIALS AND METHODS
Materials. Arrayed wells of diameter 6.96 mm, with a spacing of 9 mm, were scribed on transparencies for the experiments. Each transparency sheet was cut using a desktop cutter plotter (Wentai, JK361) which had a 90° cutting tip. The force of the needle on the cutter plotter was chosen to ensure that the transparency was not cut all the way through. The highest possible setting of 500 N was selected as the subsequent setting of 600 N pierced the transparency in some places. Furthermore, a speed of 50 mm/s was chosen so that the accuracy of the shape of the design was well preserved. This was 850
dx.doi.org/10.1021/la304394s | Langmuir 2013, 29, 849−855
Langmuir
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Article
RESULTS AND DISCUSSION We found that at low magnifications the optical profilometer gave poor results due to light−matter interaction characteristics of the surface. From a series of tests conducted, we found that higher magnification measurements made using a 50× objective lens and a 2× multiplier lens combination gave the best results. Because of limited spatial coverage, these images had to be later stitched together in order to obtain a complete depiction of the profile. A typical complete scan of the scribed surface is shown in Figure 3. It can be seen that the profile generated did not
found (≈95°) which indicated a period when the liquid body was not fully adherent on the surface despite appearing to contact. Interestingly, as more liquid is dispensed, the liquid body could then begin to develop stronger adhesion on to the surface, causing it to assume a droplike shape. In the process, the contact angle reduced until the critical advancing state for the drop. This is removed from the typical situation where contact angles increase from equilibrium to critical advancing when perturbed physically to move. We are, at this stage, not certain as to the cause of the non-fully adherent state. It is plausible that air may be trapped between the liquid and substrate during contact, which then caused the pendant drop to be deformed with increasing volume delivery to create the larger than expected contact angle. Once the drop was established on the surface, the contact line began to extend outward from the center with more liquid dispensed. We found, from numerous tests conducted, that the extension is not uniform in the radial sense but rather tended to always grow more in one direction (in our case we found faster movement toward the right, as seen in image C). This indicated that resistance to contact line advancement is almost never uniform on the surface. Once the contact line reached the scribed region of the well on the right, it became pinned at that location, allowing the remaining growing liquid body to have the left contact line moving until it too became pinned by the scribed region of the well there (D). Throughout this process, the contact angle trace remained relatively constant. We also found very little asymmetry (
