Design and fabrication of the V-Chip

The V-Chip employs SlipChip technology15,16 but introduces new flow paths and a new readout strategy. In the SlipChip, two pieces of glass etched with microfluidic wells and channels are assembled together in the presence of mineral oil. A fluidic path is formed when the two plates aligned in a specific configuration. Samples or reagents are preloaded through drilled holes using a pipette, and the top plate is then moved relative to the bottom plate to enable the diffusion and reaction of samples or reagents. Unlike previous SlipChip methods, our V-Chip employs catalase as a probe to generate oxygen, directly linking the ELISA reaction to the generation of ink-based bar charts and the direct visualization of assay results without the need for external instruments.

Two 75 × 50-mm glass slides are used to fabricate the V-Chip device. Channel patterns are first drawn with AutoCAD software and fabricated onto glass surfaces by standard photolithography and glass etching methods. A detailed protocol is provided in the Methods section and in Supplementary Fig. S1. Typical finished top and bottom plates are shown in Supplementary Fig. S2. We have designed and fabricated devices with different throughput levels, including single-, 6-, 10-, 30- and 50-plexed measurements.

Working principle of the V-Chip

As shown in Fig. 1a, the device is first placed in the loading position after assembly, where the rectangular wells of the top plate and the bottom plate partially overlap to form an ‘N’-shaped fluidic path from the left side to the right side in the horizontal direction. In this position, reagents or samples can be loaded through the drilled holes on the left of the V-Chip (Supplementary Fig. S2). Before the sample assay, the top flow lane is preloaded with ink and the second lane from the top is kept blank to serve as an air spacer, thereby avoiding direct contact between the sample and the ink. This is essential because hydrogen peroxide may bleach the ink solution and affect the visual readout of the results. The third lane is the sample lane, where the ELISA assay is carried out. This lane can also be filled with catalase molecules directly for an oxygen generation test. The bottom-most lane is preloaded with hydrogen peroxide. An oblique slide of the device causes the horizontal fluidic paths to separate and re-form into independent units arranged in parallel in the vertical direction (Fig. 1a and Supplementary Movie 1). In each vertical unit, the wells partially overlap to form a closed ‘Z’-shaped path, bringing the hydrogen peroxide into contact with the catalase probe. Instantly, oxygen is generated and the ink in the top row of wells is pushed into the small bars (Fig. 1a and Supplementary Movie 2). Because the channels are vented to atmospheric pressure, the ink bands will continue moving in the channel until the oxygen produced is insufficient to push them. To stop the movement of the ink bar for a stable reading, the glass plates can be slipped back to the original loading position, releasing the generated oxygen through the loading holes and thereby discontinuing the propulsion of the inked bars. Sliding back the glass plates separates the vertical connexions and thereby immediately stops the ink movement. The oxygen, which is released in the horizontal direction, will not cause a spill-out as the inlet/outlet holes can serve as reagent reservoirs.

Validation and optimization of V-Chip ELISA assays

Before the V-Chip ELISA assay, capture antibodies are covalently immobilized on the epoxy-terminated glass surface in the sample wells (Fig. 1b and Supplementary Fig. S3; 20). Coating with multiple capture antibodies can be achieved by microarray printing or simply by inking the wells with a sharp swab tip (Fig. 1c). The roughness of the etched glass surface in the capture antibody wells greatly increases the coating efficiency (Supplementary Fig. S4). The location of the capture antibody and the multi-well surface coating are demonstrated in Fig. 1c, in which food dyes are used to visually represent each individual antibody. After the assembly of the V-Chip, an assay sample (for example, cell lysate or patient serum) is flowed through the ELISA channel (the third lane of the V-Chip fluidic handling region). Because of the ultra-miniaturization in microfabrication technology, only a 10-μl sample is required to complete a V-Chip ELISA reaction to detect biomarkers in serum. This is a great advantage for the application of the device to a POC blood test, for which a finger prick might be sufficient and invasiveness can be dramatically reduced.

After the protein targets are captured by the V-Chip ELISA chambers via specific antibody–antigen reactions, a mixture of all the catalase-detecting probes (silica nanoparticles conjugated with detecting antibodies and catalase molecules) is applied. Each reaction well will form an independent sandwich structure in which its specific target antigen is captured. Repeated washing steps are applied to reduce the nonspecific binding in the V-Chip assay. Our scanning electron microscope (SEM) images indicate that the catalase probes efficiently bind onto the glass surface in the presence of antigen targets (Supplementary Fig. S5). At this stage, all protein targets and their corresponding amounts of catalase probes are positioned in individual wells and will not move even when V-Chip flow paths change from the ‘N’ to the ‘Z’ shape. At the readout position, the ‘Z’-shaped flow path allows the catalase probe to make contact with hydrogen peroxide, producing oxygen, which triggers the immediate movement of the inked bars. The volume of oxygen generated correlates with the amount of catalase and the target concentration; the design is therefore able to quantitate proteins based on the advancement of the inked bars.

Figure 1d shows the assembly, reagent loading and slide operation of a 50-plex V-Chip. The top plates and the bottom plates were aligned and partly overlapped to form the ‘N’ shaped lanes. During device assembly, fluorinated oil was used as a lubricant and to render the device airtight. Following sample loading, an oblique slide separated the horizontal fluidic paths and the wells re-connected in the vertical direction (Fig. 1d). In this position, the wells containing hydrogen peroxide and the wells containing catalase overlapped partially, allowing the hydrogen peroxide solution to diffuse and react with catalase.

To demonstrate that ink movement is related to catalase concentration, catalase solutions were directly loaded through the drilled holes in the V-Chip for an oxygen generation test. Figure 1e shows a 30-plex V-Chip that was loaded with reagents and samples. When a uniform catalase concentration was applied to the V-Chip, the bar charts showed uniform advancement (Fig. 1f); whereas with a gradient of catalase, created by a 3-h diffusion of catalase from an inlet on the bottom-right of the device, the frontier of the bar chart resembled a sigmoidal shape (Fig. 1g; 21). Figure 1h demonstrated another sigmoidal bar chart advancement pattern in a 50-plex V-Chip with a 6-h diffusion of catalase. The progressive increments indicate that the distance moved by each ink bar correlates to the concentration of the diffused catalase. The data shown in Fig. 1f–h clearly demonstrate the relationship between the advancement of the V-Chip bars and the catalase concentration.

One of the key requirements for POC assays is a timely and quantitative readout. Several factors may impact the V-Chip readout, including the amount of catalase, hydrogen peroxide concentration, reaction temperature and assay time. To optimize the assay, we prepared a single-channel V-Chip and tested all the above-listed conditions (Fig. 2). As shown in Fig. 2a, we directly loaded catalase molecules at concentrations ranging from 3 to 30 U into the top chamber, and loaded hydrogen peroxide at a concentration of 0.45 M to the bottom chamber. Upon vertically sliding the V-Chip, the catalase molecules were brought into contact with hydrogen peroxide and oxygen was immediately produced, causing the red ink in the centre of the V-Chip to be pushed to the readout region. The movements of the ink were recorded as movies and the time dependence of ink advancement is plotted in Fig. 2c. The rate of ink movement was positively correlated with catalase concentration, and a pseudo-linear relationship between catalase concentration and ink movement distance was observed at 1, 2 and 6 min (Fig. 2d). All three intervals show appropriate linearity; however, more discriminable ink distances are present in intervals larger than 2 min. We maintained a consistent readout time for each batch of experiments to achieve comparable quantitation.

Figure 2: Assays using a single-channel V-Chip. (a) In the loading position of the chip, H 2 O 2 , catalase and ink are loaded in the indicated wells. (b) Ink advancement in the V-Chip is pushed by oxygen generated as a result of catalase reacting with H 2 O 2 . Scale bar, 1 cm for both a and b. (c) Time-dependent ink advancement with the application of different concentrations of catalase and 0.45 M H 2 O 2 , at room temperature. The concentrations of catalase used were 3 U ml−1 (black), 4.5 U ml−1 (red), 6 U ml−1 (green), 9 U ml−1 (blue), 12 U ml−1 (magenta), 15 U ml−1 (brown-green), 24 U ml−1 (orange) and 30 U ml−1 (purple). (d) Linearity curves of the ink advancement readout, with different catalase concentrations and in different time ranges: 1 min (black), 2 min (red) and 6 min (blue). (e) Time-dependent ink advancement with the application of different concentrations of H 2 O 2 and 15 U catalase, at room temperature. The concentrations of H 2 O 2 used were 0.15 M (black), 0.3 M (red), 0.45 M (green), 0.6 M (blue), 0.75 M (purple) and 0.9 M (brown-green). (f) Ink advancement after 6 min plotted against H 2 O 2 concentration. Full size image

We also optimized the hydrogen peroxide concentration using a similar strategy (Fig. 2e). Surprisingly, the highest hydrogen peroxide concentration did not provide the furthest ink advancement, because catalase activity was inhibited when the hydrogen peroxide concentration reached a certain threshold19. On the basis of the results shown in Fig. 2f, the optimum hydrogen peroxide concentration was identified as 0.45 M for 15 U catalase at room temperature. In the absence of catalase, 0.45 M hydrogen peroxide decomposes very slowly and the generated oxygen is not able to move the inked bars. Storing the loaded device in the dark can further reduce the hydrogen peroxide decomposition.

Catalase activity is also related to reaction temperature, but the assay works at both room temperature and 37 °C. We observed that the movement of the red ink band was slightly faster at 37 °C than at room temperature (Supplementary Fig. S6). However, consistent temperature control is more difficult at 37 °C than at room temperature. To reduce the complexity and cost for POC applications, the experiment was performed at room temperature (25 °C). During the operation, the operator’s fingers should avoid touching the catalase reaction zone to minimize temperature changes.

On the basis of the results of the assay-optimizing experiments shown in Fig. 2, we identified the ideal reaction conditions: a hydrogen peroxide concentration of 0.45 M, reaction at room temperature, and a time interval of 2–6 min for the readout.

Single-channel V-Chip ELISA assay

We first tested the ELISA reaction with a human chorionic gonadotropin (hCG) protein target using a single-channel V-Chip. hCG is a pregnancy indicator and widely accepted biomarker for some types of cancer, including breast and ovarian cancer22. We prepared a single-channel V-Chip and performed repeated hCG assays using the same chip. To conveniently reuse the V-Chip, we used 2.8-μm magnetic beads conjugated with the hCG capture antibody for the ELISA reaction. The beads were held at the bottom of the ELISA well by a magnet (Fig. 3a) and replaced with new particles for a new assay with a different hCG concentration. We tested a range of hCG concentrations from 2 to 1 × 105 mIU and compared the assay results with a commercially available pregnancy test. Figure 3b presents the back-to-back 6-min assay results from the pregnancy kit and the V-Chip test. At low hCG concentrations, the pregnancy kit result showed a weak signal at 5 mIU and no signal at 2 mIU, while the V-Chip presented clear ink advancement even at the 2 mIU hCG concentration. At high concentrations, because of the hook effect23, the pregnancy test kit began to lose signal due to the binding saturation by the extra amount of the target, while the V-Chip still presented visual and quantitative results. Over the entire hCG concentration range, the V-Chip was able to visualize and quantify the target (Fig. 3c). The V-Chip also works with more complex sample matrices, such as serum and urine (Fig. 3e).

Figure 3: Detection of hCG using a single-channel V-Chip. (a) V-Chip hCG detection scheme. In the ELISA well, hCG is captured by magnetic beads functionalized with capture antibodies, and detected by catalase-conjugated silica nanoparticles functionalized with detecting antibodies. The reaction is initiated by sliding the V-Chip. (b) Comparison of the commercial pregnancy test and the V-Chip for sensing hCG in PBS buffer. (c) Time-dependent ink advancement in the presence of different concentrations of hCG: 2 mIU (black), 5 mIU (red), 10 mIU (blue), 1 × 102 mIU (cyan), 1 × 103 mIU (magenta), 1 × 104 mIU (brown) and 1 × 105 mIU (navy). (d) The hCG calibration curve corresponding to ink advancement at 6 min, with the hCG concentration varying from 2 mIU to 1 × 105 mIU. (e) Detection results of spiked hCG in PBS buffer, serum and urine. The error bars in d and e represent the s.d. of three measurements. Full size image

Multiplexed V-Chip ELISA assay

A primary concern in cancer diagnosis and prognosis is the correct classification of the patient’s disease stage. The clinical method for the identification and classification of human tumours is mostly based on immunohistochemistry and fluorescence in situ hybridization24. ELISA is an alternative25 that is frequently used in monitoring the response to therapy by measuring tissue lysates and in follow-up care of patients when measuring serum biomarkers. Despite the reduced sensitivity caused by mixed cell populations, ELISA technology is still the only reliable test for large sample sizes that has the quantitative capability to analyse biomarker expression as a continuous variable rather than a dichotomous variable25. Therefore, ELISA technology serves as the standard to establish the cutoff point and define biomarker overexpression for the selection of optimal therapy strategies.

Most tumours, including breast cancer, are highly heterogeneous26. A single biomarker measurement is not reliable for diagnosis or classification. The inclusion of more biomarkers and the use of multiplexed assays are believed to be able to increase accuracy27,28,29; however, this will increase diagnostic cost significantly. In our multiplexed V-Chip, the micro-sized channels require minimal amounts of reagent and the parallel operation reduces assay time, thereby significantly reducing assay cost compared with traditional diagnostic approaches. In contrast to traditional ELISA methods, the V-Chip device is portable, affordable and potentially user-friendly, which might be suitable for disease monitoring in resource-limited areas such as developing countries or in home blood testing. To demonstrate the multiplexed capability of the V-Chip, we generated a six-plexed chip for breast cancer cell classification. We included the oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) biomarkers in the V-Chip, and used the remaining blank bars as controls30.

To assess whether our device can accomplish ER, PR and HER2 profiling, cells from five breast cancer lines (BT-474, MCF-7, SKBR-3, SUM-159 and MDA-MB-231) were counted, directly lysed in RIPA buffer and used to validate the capability of the V-Chip assay. According to previous reports, ER, PR and HER2 are all overexpressed in BT-474 cell lines (Fig. 4a; 30). Figure 4b shows the distance moved by the red ink bands in the presence of different amounts of BT-474 cells. As the number of BT-474 cells increased, the bar distance values of the three biomarkers changed accordingly, indicating that ER, PR and HER2 are all expressed in the BT-474 cell line. The control channels never moved a significant distance larger than 0.3 V-Chip unit (Fig. 4b). The dependent increment of the distances for ER, PR and HER2 suggests that the V-Chip may be suitable for clinical use to evaluate the levels of these receptors. Using this device, we can detect these biomarkers with sample sizes as low as 1000 BT-474 cells ml−1 (Fig. 4c).

Figure 4: Multiplexed detection of biomarkers from cell lysates. (a) Fluorescent images of BT-474, SKBR-3, MCF-7, MDA-MB-231 and SUM-159 cells, stained with Cy3 (ER and PR) and FITC (HER2). Scale bar, 100 μm. (b) The top four ink advancement images show the results of assays on BT-474 lysates with increasing numbers of cells; the bottom four images show the results of the assays on SKBR-3, MCF-7, SUM-159 and MDA-MB-231 cell lysates with a concentration of 1 × 106 cells ml−1. C, E, P and H represent control, ER, PR and HER2, respectively. Scale bar, 1 cm. (c) Quantitation curves of ER, PR and HER2 in BT-474 assays. (d) V-Chip readouts of the five cell lysates at a concentration of 1 × 106 cells ml−1. The error bars in c and d represent the s.d. of three measurements. Full size image

Using a cell concentration of 106 cells ml−1, the V-Chip was next used to distinguish the biomarker expression profiles of different breast cancer cell lines (Fig. 4b, bottom). Considering the distance values in BT-474 cells to be 100%, and assuming that the distance values are proportional to biomarker expression, SKBR-3 cells expressed 92% HER2, while MCF-7 cells expressed 92% ER and 29% HER2 (Fig. 4d). However, these three biomarkers were rarely expressed in SUM-159 and MDA-MB-231 cells, which are known to have low expression of ER, PR and HER2. These results are consistent with previous reports and our own fluorescent measurements30.

Parallel measurements of multiple serum samples

Carcinoembryonic antigen (CEA) has become one of the most convincing serum biomarkers as it provides fast, non-invasive, reproducible and quantitative indications in follow-up care and when monitoring the therapy of breast cancer patients. Although CEA cannot be used to diagnose primary cancer, its applications in early-stage prediction of metastases and in monitoring the response to metastatic therapy are promising31. To assess the prognostic value of serum biomarkers, multivariate analysis of a large number of patient sera in a short follow-up period is required32. In the present work, we tested serum samples from 10 patients using the V-Chip.

Figure 5a shows a V-Chip assay image of the 10-serum test and Fig. 5b shows both the results from the clinical ELISA method (Supplementary Fig. S7 and Supplementary Table S1) and the average bar advancements of three V-Chip tests listed side-by-side. The trends of both types of tests are correlated. In the plot of V-Chip ink advancement against CEA concentration values in Fig. 5c, it is not surprising that the curve is pseudo-exponential rather than linear. The causes may include antibody–antigen binding constant, catalase catalytic activity and other related thermodynamic effects. It should be noted that the sensitivity of the V-Chip is not as good as that of highly sophisticated clinical instruments and that the V-Chip performs better at concentrations higher than 5 ng ml−1. The sensitivity of the V-Chip can certainly be improved if narrower and longer bar channels are used.