On-Site Melanoma Diagnosis Utilizing a Swellable Microneedle-Assisted Skin Interstitial Fluid Sampling and a Microfluidic Particle Dam for Visual Quantification of S100A1

Abstract

Malignant melanoma (MM) is the most aggressive form of skin cancer. The delay in treatment will induce metastasis, resulting in a poor prognosis and even death. Here, a two-step strategy for on-site diagnosis of MM is developed based on the extraction and direct visual quantification of S100A1, a biomarker for melanoma. First, a swellable microneedle is utilized to extract S100A1 in skin interstitial fluid (ISF) with minimal invasion. After elution, antibody-conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are introduced. A high expression level of S100A1 gives rise to a robust binding between MMPs and PMPs and reduces the number of free PMPs. By loading the reacted solution into the device with a microfluidic particle dam, the quantity of free PMPs after magnetic separation is displayed with their accumulation length inversely proportional to S100A1 levels. A limit of detection of 18.7 ng mL^-1 for S100A1 is achieved. The animal experiment indicates that ISF-based S100A1 quantification using the proposed strategy exhibits a significantly higher sensitivity compared with conventional serum-based detection. In addition, the result is highly comparable with the gold standard enzyme-linked immunosorbent assay based on Lin's concordance correlation coefficient, suggesting the high practicality for routine monitoring of melanoma.

Keywords: S100A1; interstitial fluid; malignant melanoma; microfluidics; swellable microneedle.

Figure 1

Illustration of a swellable microneedle patch and a self‐powered microfluidic device for melanoma diagnosis. i) Extraction of ISF with the administration of a swellable microneedle patch on the skin. ii) Elution in a centrifuge tube. iii) Reaction between S100A1 and antibody‐conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). iv) Visual quantification of S100A1 using a microfluidic device with a microfluidic particle dam. With the high concentration of S100A1, MMPs and PMPs simultaneously bind to S100A1 to form the “MMPs‐S100A1‐PMPs” structure, which lessens the number of free PMPs escaping from a magnetic separator, resulting in a short PMP accumulation length that can be visually quantified without an additional instrument. With the lower concentration of S100A1, however, a longer PMP accumulation length is observed due to the insufficient binding between MMPs and PMPs.

Figure 2

Optimization of hydrogel‐based swellable microneedle for extraction of macromolecules. a) Optical image of microneedles (left), the agarose gel before (middle) and after the insertion (right). Scale bar: 1 mm. b) Demonstration of FITC‐dextran (molecular weight: 15 kDa) extraction from the skin model. The control group (left, agarose gel without FITC‐dextran), the 10–100 kDa HA fabricated microneedle group (middle) and the 200–400 kDa HA fabricated microneedle group (right). Scale bar: 500 µm. c) Optical density at wavelength 450 nm to measure the extracted S100A1 concentration in skin model (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 100, and 200 ng mL⁻¹) using HA fabricated microneedles (n = 3). d) Subset data of Figure 2c where the concentration of S100A1 ranges from 0 – 10 ng m⁻¹L (n = 3).

Figure 3

Detection of mouse S100A1 standards on microchip. a) Schematic illustration of S100A1 detection using the microfluidic chip. b) Optimization of reaction time (n = 3). c) Optical image of accumulated PMPs. d) PMP accumulation length versus with respect to S100A1 concentrations (n = 3). e) Linear regression with S100A1 concentration ranging from 0 to 50 ng mL⁻¹. f) Selectivity against potential interfering factors (n = 3). The measured PMP accumulation length showed that only S100A1 (500 ng mL⁻¹) shortened the PMP accumulation while other interfering biomarkers with higher concentrations (S100A4, 1 µg mL⁻¹; S100A8, 250 µg mL⁻¹; S100A13, 1 µg mL⁻¹; S100B, 1 mg mL⁻¹; LDH‐A, 10 µg mL⁻¹; LDH‐B, 10 µg mL⁻¹; MIA, 1 mg mL⁻¹) did not cause any robust binding between MMPs and PMPs.

Figure 4

Standard curves for S100A1 extracted from skin model. a) Schematic illustration of S100A1 extraction using microneedle patch, followed by ELISA and microchip analyses. b) ELISA measurement for S100A1 in the skin model. c) Standard curve of ELISA measurement using non‐linear fitting d) PMP accumulation length in microfluidic chip against S100A1 concentrations in the skin model. e) Standard curve of microchip measurement using the non‐linear fitting. All experiments were repeated twice.

Figure 5

Biocompatibility of the swellable microneedles. a) The cell viability test via Alamar blue staining (n = 3). b) Images of live and dead assay of MSC viability treated with blank, 100 kDa HA, 100 kDa MeHA, and 200–400 kDa MeHA fabricated microneedles. Scale bar: 100 µm c) Optical images of mice skin before and 1, 3, 5, and 15 min after microneedle penetration. Scale bar: 1 mm d) The H&E staining of the mouse skin after microneedle penetration. Scale bar: 100 µm.

Figure 6

In vivo model of melanoma. a–e) Mice with different tumor sizes. Scale bar: 1 mm. f) Microneedle patch administration. Scale bar: 1 mm. g) H&E staining of melanoma tissue and IHC staining of S100A1. Scale bar (H&E staining): 500 µm. Scale bar (IHC): 50 µm. h) IHC staining of S100A1 for tumors with different sizes. Scale bar: 50 µm.

Figure 7

Quantitative measurement of S100A1 based on ELISA and microfluidic chip. a) ELISA measurement of S100A1 extracted from ISF and serum. b) Original concentration of S100A1 in ISF and serum based on the inverse regression. c) Relationship between the tumor size and S100A1 expression level. d) Microchip measurement of S100A1 extracted from ISF (n = 3). e) Original concentration of S100A1 in ISF based on the inverse regression (n = 3). f) Comparison of S100A1 expression levels measured by mouse S100A1 ELISA kit (y‐axis) and microfluidic chips (x‐axis). The correlation was determined on the basis of Lin's concordance correlation coefficient ${\widehat{\rho }}_c$ = 0.916, validating a moderate agreement between the gold standard ELISA and the proposed microfluidic chip.