Paper-Based Microfluidic Device with Upconversion Fluorescence

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Letter pubs.acs.org/ac

Paper-Based Microfluidic Device with Upconversion Fluorescence Assay Mengyuan He and Zhihong Liu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, China S Supporting Information *

ABSTRACT: A paper-based microfluidic device with upconversion fluorescence assay (named as UC-μPAD) is proposed. The device is fabricated on a normal office printing sheet with a simple plotting method. Upconversion phosphors (UCPs) tagged with specific probes are spotted to the test zones on the μPAD, followed by the introduction of assay targets. Upconversion fluorescence measurements are directly conducted on the test zones after the completion of the probe-to-target reactions, without any posttreatments. The UC-μPAD features very easy fabrication and operation, simple and fast detection, low cost, and high sensitivity. UC-μPAD is a promising prospect for a clinical point-of-care test.

T

eration of the high complexity of both clinical samples and paper substrates, upconversion fluorescence would be particularly suitable for μPAD-based clinical analytics. Therefore, the combination of μPAD with upconversion fluorescence assay is likely to afford a promising tool for a point-of-care test (POCT) with expected simplicity, accuracy, and sensitivity, promoting the application of μPAD in clinical diagnosis. The UC-μPAD was patterned on a piece of normal office printing paper with a simple plotting method according to the literature (Figure S1, Supporting Information).8 In brief, we first printed a specific pattern on the printing sheet and made a metal template in accordance with the shape of the pattern. Next, we plotted the pattern on the paper according to a template with a permanent marker and left the resultant patterned paper at room temperature to evaporate the solvent. The resins remaining in the marks on the paper thus formed the hydrophobic barriers and produced circular detection regions (test zones). The diameter of the detection region was 4 mm, requiring ca. 4 μL of solution to fill with uniformity (Figure S2, Supporting Information), so that the amount of reagents and samples was 1−2 orders of magnitude lower than that of conventional liquid detection methods (tens to hundreds of microliters). The signal was measured with a simple homemade setup for upconversion fluorescence (Figure S3, Supporting Information). Briefly, the paper loaded with reagents and samples in the test zones was stuck on the surface of a solid sample holder followed by recording the fluorescene intensity at a selected wavelength (547 nm for our UCPs) with

he microfluidic paper-based analytical device (μPAD) is a potential point-of-care diagnostic technology put forward by Whitesides and co-workers in 2007.1 Thereafter, increasing attention has been paid to μPAD because of its attractive features including low cost, ease of use (without the need of an external fluid-driving facility), low consumption of reagents and samples, and so on.2 In the last several years, different kinds of μPAD have been exploited in the detection of species ranging from small molecules to biological macromolecules.3 Up to now, colorimetric detection is the most commonly used assay method on μPAD owing to the easy operation and straightforward signal readout, but the practical use of this method is restricted by its relatively low sensitivity.4 The fluorescence assay is another kind of optical method possessing inherently much higher sensitivity than colorimetric methods. However, it is very difficult to perform fluorometric measurements directly on μPAD due to the rather serious background fluorescence of additives and scattering light in the paper substrate, which leads to the extremely demanding requirements of paper types.5 As such, it is still highly desired to develop μPAD with high sensitivity to realize the assay of lowconcentration components in clinical samples. To meet the challenge, we made our efforts to embed upconversion fluorescence assay in μPAD, which we named as UC-μPAD in the present work. Upconversion fluorescence is a type of anti-Stokes luminescence emitted from upconversion phosphors (UCPs) when they are excited with near-infrared (NIR) light.6 Previous studies have shown the strong ability of UCPs to circumvent the problem of autofluorescence and/or scattering light because of the NIR-excitation nature, which considerably improves the robustness and sensitivity of fluorescence assays.7 In consid© 2013 American Chemical Society

Received: November 14, 2013 Accepted: December 3, 2013 Published: December 3, 2013 11691

dx.doi.org/10.1021/ac403693g | Anal. Chem. 2013, 85, 11691−11694

Analytical Chemistry

Letter

target quantification. The overall quenching of UCPs fluorescence by the dye tagged to the peptide reached a level of 90.2%, and only 9.3% quenching was observed when free TAMRA was directly added to UCPs on the test zone (Figure S7, Supporting Information), indicating that the bridging of the peptide was essential for the RET process. The photostability of the UCPs−peptide−TAMRA complex was also examined. As seen, even under continuous illumination with NIR (980 nm) light for up to one hour, the fluorescence intensity remained almost unchanged (Figure S8, Supporting Information). In a typical run for the target detection, the UCPs−peptide− TAMRA complex was first spotted on the test zones and allowed to air-dry under ambient condition. Aqueous buffer solutions contaning varying amounts of MMP-2 were then dropped on the zones initiating the cleavage of the peptide substrate. Because RET is a process sharply dependent on the distance between the fluorophore and the quencher, the energy transfer is blocked once the peptide is cut off by the enzyme. As such, the signals of the reactions were directly measured without any separation steps, which remarkably simplified the procedure and shortened the assay time. With an increase in the concentration of MMP-2, a gradual increase of UCPs fluorescence was observed (Figure 1a). A control experiment

a 980 nm CW laser as the light source. Note that the setup has no need of an excitation spectrometer because a laser with fixed wavelength is used for excitation. After the device fabrication, water-soluble UCPs, NaYF4:Yb,Er nanocrystals with dendritic shape (Figure S4, Supporting Information), were synthesized using our reported routes. 9 The UCPs surface was modified with poly(ethylenimine) (PEI) for subsequent labeling of assay probes. Next, we examined the stability of the fluorescence signal on the device. To the test zone, 4 μL of UCPs aqueous solution was spotted with the top and bottom faces of the test zone open to the atmosphere. After the evaporation of most of the solvent on the surface, the fluorescence intensity of the test zone was detected. The signal intensity of UCPs on the paper remained unchanged for more than 96 h when kept in the atmosphere (Figure S5a, Supporting Information). We then tried washing the UCPs by adding water to the top of the zone while having its bottom in contact with filter paper which absorbed the water. After five cycles of washing, the fluorescence intensity of UCPs in the test zone showed no obvious decrease (Figure S5b, Supporting Information). The above results proved that UCPs could firmly adhere to the paper surface presumably due to physical adsorption forces, which ensured the signal stability in subsequent sampling and detection processes. It is worth noting that the paper matrix exhibited completely zero-background with 980 nm excitation, revealing the merit of upconversion fluorescence. To verify the feasibility of using this UC-μPAD for a biomolecule assay, we selected matrix metalloproteinase-2 (MMP-2), an important biomarker in blood as the proof-ofconcept target. Studies have shown that almost every type of human cancer, diabetes, and hypertension has a close relationship with MMP-2, with its concentration in blood increasing to varying degrees. The level of MMP-2 in healthy human serum ranges from 500 to 700 ng/mL. The assay principle is schematically shown in Scheme 1. A peptide

Figure 1. (a) Upconversion fluorescence of the test zones with various concentrations of MMP-2 (0, 50, 500, 800, 1000, 2000, and 5000 pg/ mL). (b) Linear dependence of the relative fluorescence intensity (F − F0)/F0 on the concentration of MMP-2 within the range of 50−5000 pg/mL. Data are presented as the average ± SD from three independent measurements.

Scheme 1. Schematic Illustration of the UC-μPAD for the MMP-2 Assay Based on the Cleavage of a Specific Peptide Substrate by the Target and the Resonance Energy Transfer from Upconversion Phosphors to TAMRA Dye

showed that the protein (MMP-2) itself did not affect the fluorescence of UCPs (Figure S9, Supporting Information). Within the range of 50−5000 pg/mL, a linear calibration with a correlation coefficient of 0.992 was obtained between the target concentration and the fluorescence recovery, (F − F0)/F0, where F and F0 were the fluorescence intensities in the presence and in the absence of MMP-2, respectively (Figure 1b). The detection limit was determined to be 8.3 pg/mL, which was calculated as the concentration corresponding to 3 times of the standard deviation of the background signal (n = 7). Already with the above-mentioned exemption of separation steps in hand, we further investigated the incubation time needed for the enzymatic cleavage, considering that the assay time is a key factor for a device designed for potential POCT applications. Taking 1 ng/mL MMP-2 as an example, we measured the fluorescence of the UC-μPAD as a function of the incubation time. The fluorescence intensity reached the maximum value within a 30 min reaction (Figure S10, Supporting Information). While in a solution system, as is known, the same cleavage reaction normally requires more than 120 min to reach the equilibrium.9−11 Similar to other reported μPADs, we believe that the high surface-to-volume ratio, porous structure, and the ultrasmall volume of the paper

substrate specific for MMP-2 was covalently linked to the UCPs surface, and the successful conjugation was characterized by UV−vis spectra (Figure S6, Supporting Information). A tetramethylrhodamine (TAMRA) dye was tagged at the end of the peptide, which quenched the fluorescence of UCPs due to the resonance energy transfer (RET) from UCPs to the dye. The emission of UCPs is expected to be restored in response to the cleavage of the peptide by MMP-2,10 which is the basis for 11692

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Analytical Chemistry

Letter

devices are the reasons for the dramatic reduction in the reaction time. Before we moved on to the target assay in clinical samples, we looked into the specificity of the proposed device toward MMP-2. In the test zones containing the UCPs−peptide− TAMRA complex, various disruptors including biomolecules and metal ions were introduced in place of MMP-2 following an identical assay procedure. None of the analyzed species including MMP-1, another member of the matrix metalloproteinase family, caused obvious signal change even when their concentrations were 5 times higher than that of MMP-2 (Figure 2).

Table 1. Determination of the MMP-2 Level in Human Serum and Whole Blood Samples Using the UC-μPAD samplea

measured (pg/ mL)

added (pg/ mL)

found (pg/ mL)

recovery (%)

RSD (%)

1 2 3 4 5 6 7 8 9 10

575.9 685.0 449.0 681.5 644.5 582.5 675.7 636.7 454.6 671.8

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000

1525.0 1559.5 1516.7 1576.4 1690.8 1494.1 1707.6 1557.4 1494.1 1649.5

94.9 87.5 106.8 89.5 104.6 91.2 103.2 92.1 103.9 97.8

3.03 2.99 9.28 5.18 3.45 0.32 8.79 3.94 1.59 3.85

a Samples 1−5 were human serum, and samples 6−10 were whole blood.

established DNA aptamer with high affinity.13 The assay of CEA was principally based on the conformation transition of the aptamer induced by the interaction with target, which altered the RET efficiency between UCPs and the quencher, as illustrated in Scheme 2. The aptamer (please see Supporting Scheme 2. Illustration of the UC-μPAD for CEA Detection Based on the Recognition of CEA Aptamer by the Target and the Fluorescence Quenching of Upconversion Phosphors by Graphene Oxide Figure 2. The relative fluorescence intensity of test zones added with UCPs−peptide−TAMRA plus different substances. The concentration of MMP-2 was 200 pg/mL and that of other interfering species was 1000 pg/mL. Blank represents the zone added with only UCPs− peptide−TAMRA. Data are presented as the average ± SD from three independent measurements. All species were dissolved in TCNB buffer solution.

With the specificity verified, we validated the applicability of this UC-μPAD by testing the recovery rate in spiked blood samples. Taking into account the normal MMP-2 level in healthy human blood as well as the linear range of our method, the clinical samples were 1000-fold diluted with buffer. That is to say, indeed only 4 nL of blood is needed for a single test. Considering that the slope of the calibration curve in blood samples could be slightly different from that in aqueous buffer, we first acquired a calibration curve in a diluted serum by adding certain amounts of external MMP-2 and subtracting the signal contributed by internal MMP-2 (Figure S11, Supporting Information). We altogether analyzed ten individual real samples including five human serum samples and five whole blood samples, and the results are presented in Table 1. The detected MMP-2 levels in blood samples are in the range of 450−700 ng/mL (taking the 1000-fold dilution into account), which are consistent with the reported level obtained with the ELISA method.12 The recovery rates of spiked blood samples ranged from 87.5% to 106.8% with relative standard deviations (RSD) of less than 10%, which shows the practicability of the UC-μPAD in real samples. In addition to the above substrate-cleavage model for protein enzyme assay, we further constructed another UC-μPAD based on aptamer−protein recognition for carcinoembryonic antigen (CEA), in order to demonstrate the generality of our concept. CEA is also a biomarker in blood for diseases, which has an

Information for the sequence) was covalently linked to UCPs as the probe, which was also confirmed by UV−vis characterization (Figure S12, Supporting Information). In this case, poly(acrylic acid) (PAA) modified NaYF4:Yb,Er nanoparticles with an average size of 50 nm were used as the fluorophore,7d and graphene oxide (GO) was used as the quencher so that the labeling of the quencher to the probe could be omitted. The UCPs−aptamer conjugate was spotted on the test zone and left to dry at room temperature. After the addition of GO, the fluorescence of UCPs was quenched as a consequence of the π−π stacking of the DNA aptamer on the GO surface. The quenching was in a GO concentration-dependent manner, and the maximum quenching efficiency was ca. 94.6% (Figure 3a), which was achieved within 10 min (Figure 3b). In the presence of the target CEA, the stronger interaction between CEA and the aptamer weakened the adsorption of the aptamer on GO, resulting in restoration of the fluorescence of UCPs (Figure 3c). It is also a quick process that reached equilibrium within 45 min (Figure 3d). These results suggested that the UC-μPAD 11693

dx.doi.org/10.1021/ac403693g | Anal. Chem. 2013, 85, 11691−11694

Analytical Chemistry

Letter

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21075094, 21375098), the National Basic Research Program of China (973 Program, No. 2011CB933600), and the Program for New Century Excellent Talents in University (NCET-110402).

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Figure 3. (a) Fluorescence quenching of UCPs−aptamer (0.05 mg/ mL) by graphene oxide (GO) with the concentration ranging from 0 to 0.5 mg/mL. Inset: the fluorescence intensity of UCPs versus GO concentration. (b) Fluorescence quenching of UCPs−aptamer (0.05 mg/mL) by 0.3 mg/mL GO as a function of time. (c) Upconversion fluorescence spectra of the test zones with the introduction of various concentrations of CEA (0, 0.05, 1, 5, 50, 100, and 500 ng/mL). Inset: relative fluorescence intensity (F − F0)/F0 versus the concentration of CEA within the range of 0−500 ng/mL. Data are presented as the average ± SD from three independent measurements. (d) Time course of the fluorescence recovery with 0.3 mg/mL GO and 80 ng/mL CEA.

could be extended to the assay of diverse targets with different sensing principles. In summary, we have constructed microfluidic paper-based analytical devices with an upconversion fluorescence assay, namely, UC-μPAD, for the first time. The features of NIR excitation and anti-Stokes emission of upconversion fluorescence perfectly match the requirements of μPAD in clinical analysis. The proposed UC-μPAD offers several notable merits: (1) it is cheap: the paper device can be simply fabricated with normal office printing sheets; (2) it is rapid and extremely simple: an entire assay can be completed in less than one hour with no need of washing; (3) it requires a tiny amount of samples and reagents; (4) it is highly sensitive: the detection limit is as low as pg/mL magnitude, which is the level of lowabundance proteins in blood. Note that we did not optimize either the assay conditions or the parameters of the device since we were primarily aiming to elucidate the feasibility of this new technique, which means there are still improvements to be made to the performance. Our efforts suggest that UC-μPAD could be a competent alternative for the development of simple, rapid, inexpensive, and portable clinical diagnostic technology that may find wide applications.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details, instrumentation, the TEM characterization of UCPs, and control experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-27-8721-7886. Fax: 8627-6875-4067. 11694

dx.doi.org/10.1021/ac403693g | Anal. Chem. 2013, 85, 11691−11694