Microneedle Patch for In Situ Neutrophil Monitoring

Abstract

Monitoring neutrophil levels plays an essential role in diagnosing infections and various diseases, as well as for managing specific treatments such as chemotherapy and radiotherapy. However, the traditional complete blood count technique is invasive and challenging for quick and frequent monitoring of neutrophil. Herein, a button-like microneedle patch for rapid and convenient detection of neutrophil levels is reported without vascular invasion. This patch consists of a hollow microneedle array with a vacuum chamber for extraction of interstitial fluid, a hydrogel sheet for colorimetric detection of neutrophil level based on neutrophil-specific enzyme (myeloperoxidase), and a mobile phone application for data recording and quantification. The detection ability is validated both in vitro and in vivo in a chemotherapy-induced neutropenic rat model. Moreover, this button patch achieves precise measurement of the neutrophil levels in patients' urine and abdominal drainage fluid. This minimally invasive colorimetric patch can potentiate in situ neutrophil monitoring without blood sampling procedures or in-lab apparatus, which can serve as a substitution for routine blood counts especially for patients in less developed areas.

Keywords: cancer; microneedle; neutrophil monitoring; non‐invasive detection; portable device.

Figure 1

Schematic and characterization of the button patch for neutrophil detection. A) Schematic of the patch. After application onto the skin, blood neutrophils are locally recruited and release their specific myeloperoxidase into the ISF, which can be extracted onto a lyophilized hydrogel sheet for color development after the removal of the cap. Blood neutrophil levels can be understood by observing color development, and the data can be quantified through a mobile phone application. B) Structure of the patch. C) Photographs of side, top, and bottom views of the device. Scale bar: 3000 µm. D, E) Scanning electron microscopy images of the microneedle array. Scale bar: 500 µm. F) Comparison of the amount of ISF extracted by vacuum chambers of different heights (n = 3). G) Extraction profiles of 3 mm vacuum chamber (n = 3). In F and G, data are presented as mean ± SD.

Figure 2

Characterizations of the lyophilized hydrogel sheet. A) Scanning electron microscopy images of the double‐layered hydrogel sheet. Left scale bar: 200 µm. Right scale bar: 20 µm. B) Fluorescence microscopy images of the cross‐section of the hydrogel sheet containing Cy5‐labeled GOx and FITC in represents of ABTS and mPD. Scale bar: 1000 µm. C) Up: Photographs showing the color changes of the hydrogel sheet after MPO solution (1‐50 nM) absorption for 5 minutes. Scale bar: 1000 µm. Down: RGB mean values of the circular observation window in the taken picture (n = 3). D) Up: Photographs showing the color changes of the hydrogel sheet after PMA‐stimulated neutrophil cultures (1‐16 ×10⁶/mL) absorption for 5 minutes. Scale bar: 1000 µm. Down: RGB mean values of the circular observation window in the taken picture (n = 3). E–G) Color development profile of the hydrogel sheet added with different concentrations (low: 5 nM, middle: 20 nM, high: 50 nM) of MPO solution (n = 3). H–J) RGB mean values of the photograph of hydrogel sheet added with different concentrations (low: 5 nM, middle: 20 nM, high: 50 nM) of MPO solution at different volumes (n = 3). K–M) RGB mean values of the photograph of hydrogel sheet added with different concentrations (low: 5 nM, middle: 20 nM, high: 50 nM) of MPO solution with the presence of 5 mM H₂O₂ and 20 mM glucose (n = 3). N–P) RGB mean values of the photograph of hydrogel sheet added with different concentrations (low: 5 nM, middle: 20 nM, high: 50 nM) of MPO solution at different temperatures (n = 3). In C‐P, data are presented as mean ± SD. In H‐J, statistical significance was determined by the two‐tailed Student's t‐test. In K‐P, statistical significance was determined by the one‐way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Comparison of MPO and neutrophil levels of untreated and neutropenia rats. A) Schematic of the induction of rat neutropenia model and the timing of detection. B) Comparison of blood neutrophil levels of untreated and neutropenia rats at day 5 (n = 5). C) ISF MPO level of untreated and neutropenia rats at different time points after the onset of inflammatory stimulation (n = 3). D) Comparison of ISF MPO level at 2 hours after the onset of stimulation (n = 3). E) Representative hematoxylin and eosin (H&E) stained sections of rat skin before and after pro‐inflammation. Scale bar: 1 mm. F) Quantification of skin neutrophil level from H&E stained sections of rat skin (n = 3). G) Comparison of skin neutrophil level at 2 hours after the onset of stimulation (n = 3). H) Photographs of the hydrogel sheet within the observation window after 2 hours of patch application with PMA and 5 minutes of ISF extraction. Scale bar: 1000 µm. I) RGB mean values of the circular observation window in the picture in H) (n = 5). J–L, Scatter plot showing the colorimetric results on each rat corresponding to blood neutrophil levels (n = 10). In B–D, F, G, and I, data are presented as mean ± SD. Statistical significance was determined by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Clinical translation potential of the button patch. A, B) Neutrophil counts of patient's urine A) and abdominal drainage fluid B) samples with corresponding color developed images. Scale bar: 1000 µm. And linear regression of the red, green, and blue values against neutrophil concentration in human abdominal drainage fluid. C) Demonstration of the software interface and illustration of the application potential of the patch clinical body fluid samples. D) Demonstration of the patch application (left) and photographs of human skin recovery at different times after patch removal (right). Left scale bar: 1 cm. Right Scale bar: 3000 µm. In A and B, data are presented as mean ± SD.