Squeezed flow preconcentration for probe tip biosensors

Analytical Biochemistry 444 (2014) 57–59

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Squeezed flow preconcentration for probe tip biosensors Brandon Huey-Ping Cheong a, Oi Wah Liew b, Tuck Wah Ng a,⇑ a

Laboratory for Optics and Applied Mechanics, Department of Mechanical and Aerospace Engineering, Monash University, Clayton, Victoria 3800, Australia Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Centre for Translational Medicine, Singapore 117599, Singapore b

a r t i c l e

i n f o

Article history: Received 18 July 2013 Received in revised form 27 September 2013 Accepted 2 October 2013 Available online 10 October 2013 Keywords: Preconcentration Probe tips Squeeze flow Green fluorescent protein

a b s t r a c t The preconcentration of analytes improves sensing using probe tips. In this work, we report a method based on creating a squeeze flow between a cylinder and circular coverslip to preconcentrate material at the liquid–gas interface while allowing a probe tip to be readily inserted there. In verification tests using enhanced green fluorescent protein, this capacity is proven. We estimated a 9.7 times increase in probability for fluorophores to be picked up at the tip using inference from fluorescence intensity distributions found. The method is expeditious, simple, and inexpensive, and it does not require any electrical energy source to operate. Ó 2013 Elsevier Inc. All rights reserved.

For biosensors, the probe tip is arguably the most popular architecture to date due to its ability to sample from a small spatial location. Since the development of the scanning probe microscope (SPM)1, considerable effort has been made to functionalize the tips of cantilevers to operate as biosensing probes [1]. These probes, however, do not need to operate within the constraints of an SPM. Nanopipettes have been reported to function as nonlabel biosensing probes [2], whereas a more recent innovation of shaping a nanobeam photonic crystal (PC) cavity into a nanoprobe offers the feature of not requiring any analyte drawn [3]. Much of the efforts expended on biosensors based on probe tips have been to imbue them with increased sensitivity. Nevertheless, this can lead to higher fabrication costs. This demand for sensitivity can be logically ameliorated if the probe samples from an analyte that is preconcentrated. The preconcentration of analytes is acknowledged to be an important procedure in biochemical analysis, particularly when small liquid volumes are used. The advantages offered include an increase in signal-to-noise ratios during sensing and the ability to detect for trace species. Sample preconcentration methods can be broadly classified as surface binding, electrokinetic equilibrium based, or membrane based. Solid phase extraction [4,5] is arguably the most well-known surface binding approach. Electrokinetic equilibrium methods include isotachophoresis [6,7], isoelectric focusing [8], and temperature gradient focusing [9,10], whereas ⇑ Corresponding author. E-mail address: [email protected] (T.W. Ng). Abbreviations used: SPM, scanning probe microscope; EGFP, enhanced green fluorescent protein. 1

0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.10.002

membrane-based approaches comprise electrophoretic filtering [11,12], evaporation [13], and concentration polarization [14]. Techniques that operate without the need for any energy source make it possible for biochemical analyses to be performed in resource-limited laboratories in tandem with novel fluid handling implements [15–17]. A method based on drop evaporation has been shown to increase the surface area exposure to air [18,19] for maximal evaporation and to ensure no further increases once specified analyte concentrations have been achieved [19]. It also eschewed the need for intervening membranes in which residues of the analyte may be trapped there. A limitation with the approach, however, lies with the relatively long period of time needed and some loss in material viability. A method of collecting samples near the liquid–gas interface to facilitate high content microscopy through squeezing a drop was recently demonstrated [20]. In the current work, we adapt the method to preconcentrate samples in order to improve probe tip biosensing capabilities. With this approach (see Fig. 1A), a sample drop is pipetted onto the center of a cylinder. A circular coverslip, of the same diameter as the cylinder, is then placed on top of the sample to sandwich the sample. From the ensuing squeeze flow, a high proportion of the material contained in the sample will collect at the liquid–air interface. Although the height of the sandwiched liquid film is low, the matching diameter of the coverslip and cylinder allows a probe tip to be inserted into it such that concentrated material at the liquid– air interface can be collected (see Fig. 1B). The experimental sample used was enhanced green fluorescent protein (EGFP), isolated from genetically modified Escherichia coli, and purified by chromatography. After elution of the proteins from

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Squeezed flow preconcentration for probe tip biosensors / B.Huey-Ping Cheong et al. / Anal. Biochem. 444 (2014) 57–59

Fig.1. Schematic description of the preconcentration method to improve probe tip biosensing. A sample drop is placed on a cylinder (A) in which a circular coverslip is placed on top, causing material within the sample to predominantly collect at the liquid–air interface of the squeezed drop. By inserting the probe tip in between the coverslip and cylinder, and within the interface region (B), it is possible to collect concentrated amounts of the sample through capillary action. The graph in panel C shows the fluorescent concentration amplification (FA) expected using an EGFP sample by inserting a probe tip as a function of a. In the inset of panel C (also plotted using the same axis parameters), it can be seen that this amplification is increased close to 9.7 times with a = 0.99. The probe tip in panel D (imaged in brightfield), when inserted into the EGFP sample without preconcentration shows the fluorescence image displayed in panel E, whereas with preconcentration it has the image displayed in panel F.

the chromatographic matrix, the sample was then dialyzed into Tris–Cl (pH 8.0), checked for purity by SDS–PAGE, and quantified using the BCA (bisinchoninic acid) protein assay (Pierce, USA). The circular coverslip used was made of borosilicate glass and had a diameter and thickness of 13 mm and 0.13–0.17 mm, respectively. The cylinder, with a diameter of 13 mm and a height of 20 mm, was machined out of aluminum. The flat surface, on which the sample was to be deposited, was polished to prevent trapping of material during the squeezing flow. The edges of this surface were also slightly beveled to permit easier insertion of the probe tip. The probe tip used (Elgiloy, MS301G) had a rated diameter of 3–4 lm. In all of the experiments, the volume dispensed (using an Eppendorf manual pipette) was kept at 4 ll. To maintain a constant thickness of the squeezed liquid sample, the coverslip was attached to a motorized translator (Zaber, T-LS13M) driven by a computer in which its speed and position of descent onto the sample below could be controlled. The preconcentration capability at the edge region was evaluated by measuring the intensity distribution with an inverted fluorescence microscope (Olympus, IX81) with a 20 objective. The improved probability of samples at the preconcentrated region to attach to the probe tip was also verified using fluorescence. The number of fluorophores at a particular location is proportional to intensity. If we consider the fluorescence intensity distribution I(r) at radial distance r from the center of the coverslip, the intensity sum from radial distance a to R (radius of coverslip) is given by:

Pða;RÞ ¼ 2p

Z

r¼R

rIðrÞdr:

ð1Þ

r¼a

Suppose that the thickness of the liquid chamber is h. The volume included from radial distance a to R is given by:

V ða;RÞ ¼ phðR2  a2 Þ:

ð2Þ

Consequently, the amplification in concentration in the volume region covered from radial distance a to R over the entire volume of the liquid in the chamber is given by:

R r¼R

FAða;RÞ ¼

R2 r¼a rIðrÞdr Pða;RÞ V ð0;RÞ ¼ 2 : R Pð0;RÞ V ða;RÞ ðR  a2 Þ r¼R rIðrÞdr r¼0

ð3Þ

With the process conducted five times using a resolution sampling at 50-lm intervals, the standard deviation over the five runs was limited to 15% of the mean for 200 lm from the coverslip edge (see Fig. S1 in online Supplementary material). This demonstrates good repeatability. A fluorescence intensity versus radial position distribution (by normalizing R to 1) obtained by the preconcentrated process was experimentally found to be approximated by the following functions: IðrÞ ¼ 1:304r5  3:011r 4 þ 2:28r3  0:5467r 2 þ 0:058r þ 0:019 r < 0:96 : 0:96 6 r 6 1 IðrÞ ¼ 246:09r2  461:34r þ 216:05 ð4Þ

Squeezed flow preconcentration for probe tip biosensors / B.Huey-Ping Cheong et al. / Anal. Biochem. 444 (2014) 57–59

With these functions input into Eq. (3), the amplification in concentration is plotted in Fig. 1C. By setting the value of a as close as possible to 1 (the edge of the coverslip), it can be seen that the probability of picking a fluorophore increases considerably. In fact, with a = 0.99, which translates to inserting the tip to within 65 lm past the coverslip edge, a close to 9.7 times increase in probability in picking a fluorophore can be achieved. Fig. 1D shows the probe tip imaged using brightfield imaging. When inserted into the EGFP sample without preconcentration, the fluorescence image (Fig. 1E) has hardly any signal. When the probe tip is inserted into the preconcentration region in the gap between the coverslip and cylinder, alternatively, the fluorescence image has significant signal (Fig. 1F). This shows that the preconcentration approach of up to 9.7 times offers better ability for the probe tip to pick up fluorophores. In the practical application of this method, it is important to ensure that the volume dispensed fills exactly to the edge when squeezed. With insufficient volumes, it will be challenging to insert the probe tip deep into the coverslip and cylinder gap to collect the preconcentrated material. With excessive volumes, the sample will ooze out past the edges, which cursorily may allow for more convenient placement of the probe tip. From experiments conducted, however, we found these collected samples to be preconcentrated only between 1 and 3 times. Although a definite explanation escapes us at this moment, it is seemingly attributed to an enhanced diffusion process occurring at the edge region when liquid expanded out from the capillary cavity into open space. The steps of placing the drop on the cylinder, applying the coverslip, and inserting the probe tip into the preconcentrated region could be done rapidly (<30 s). Compared with closed channel microfluidic systems, the approach here overcomes the significant problem of clogging and is much more simple and inexpensive to implement. In summary, we have demonstrated a simple approach here based on a squeeze flow effect to preconcentrate samples for probe tip sensing. In verifying the approach using EGFP, we found that the probability of picking up fluorophores (inferred by the fluorescence intensity distribution) increased by a factor of 6 if inserted within 65 lm past the coverslip edge. The approach was experimentally verified and found to be simple and inexpensive, and it does not require any electrical sources to conduct. It is expected to be especially useful in field applications such as the monitoring of pesticide residues or organic contaminants in wastewater and/ or natural water systems [21,22], where the preconcentration process increases the detection capability of enzymes or antibodies when using probe tip biosensors.

Acknowledgments This work was supported by the Australian Research Council (Grant DP120100583). T.W.N. appreciates discussions with R. Boysen and M. Hearn at the ARC Centre for Green Chemistry.

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