Synergistically enhanced colorimetric molecular detection using smart cup: a case for instrument-free HPV-associated cancer screening

Abstract

Rationale: Early and accurate detection of disease is crucial for its prevention, identification, and treatment. However, most of disease diagnostics is still limited in clinical laboratories due to the need of complicated instruments and professional personnel. Herein, we reported a smartphone-based synergistically enhanced colorimetric method for molecular diagnostics in our point of care (POC) smart cup platform. Methods: A disposable microfluidic chip was developed for colorimetric loop-mediated isothermal amplification (LAMP) detection of multiple HPV DNA in our POC smart cup platform. The colorimetric detection takes advantage of synergistic effect of PPi^4- and H⁺ ions, two byproducts of LAMP reaction. Color signal of LAMP assay was recorded and analyzed by our custom Android app (dubbed "Hue Analyzer"). Results: Our method not only significantly improves colorimetric readout, but also provides a 10-fold increase in detection sensitivity. It has been successfully applied for HPV-associated cancer screening with spiked saliva and clinical swab samples. Conclusion: The proposed POC diagnostic platform is completely compatible with other nucleic acid biomarkers and has great potential for personalized health monitoring and disease prevention.

Keywords: HPV-associated cancer screening; hue based colorimetric detection; point-of-care testing; synergistic enhancing effect.

Figure 1

Synergistically enhanced colorimetric detection strategy of LAMP assay in non-buffered reaction solution. (A) Detection mechanism. (B) Colorimetric detection and hue value analysis of HNB indicator with 4, 6 and 8 mM Mg²⁺ at a constant pH value (8.8) and various pH values (8.8 to 6.8), respectively. (C) Colorimetric LAMP detection of HPV 16 DNA and hue value analysis in non-buffered LAMP solution and NEB LAMP reaction buffer, respectively. Error bars denote s.d. (n=3).

Figure 2

Comparison of smartphone-based hue value analysis method and traditional absorption spectrometry in colorimetric detection. (A) Custom app interface. (B) Optical image of tubes containing 120 µM HNB indicator and various Mg²⁺ ion concentrations. (C) Hue values of HNB indicator as a function of various concentrations of Mg²⁺ ions. (D) Absorption spectra of HNB with various Mg²⁺ ion concentrations. (E) Absorbance peak of HNB indicator at 650 nm as a function of various Mg²⁺ ion concentrations. Error bars denote s.d. (n=3).

Figure 3

HPV DNA detection in saliva samples in our POC diagnostic platform. (A) Photographs of four-chamber microfluidic chip (top) and smart cup equipped with smartphone (bottom). (B) Screenshots of the app interfaces: imaging/ parameter setting (left) and hue value quantitative readout (right). (C) Detection of saliva samples spiked 10³ copies of HPV 16, 18 and 31, respectively. *** indicates a significant difference in the hue value (p < 0.001, t-test). Error bars denote s.d. (n=3).

Figure 4

Cervical cancer screening with clinical swab samples in our POC diagnostic platform. (A) Screening workflow. (B) Detection results of our smart cup platform. Red dash box indicates positive. (C) Pap smear results (HSIL: High-Grade Squamous Intraepithelial Lesion; NILM: Negative for Intraepithelial Lesion or Malignancy).