Microbubbling Digital Assay

Microbubbling assay cartoonBlowing bubbles: Labeling target proteins with microbubbles enables the highly sensitive detection of protein biomarkers (femtomolar level) in a digital assay at the point-of-care. The microbubbling readout can be detected using a smartphone camera and analyzed using a smartphone application for automated image analysis, developed using machine learning.

We introduce a simple microbubbling signaling strategy and describe a smart phone-based immunoassay platform to meet the clinical need of highly sensitive biomarker quantitation at the point-of-care (POC). In the microbubbling POC assay, protein molecules are facilely detected via digital microbubbling at femtomolar level, over 160 times more sensitive than current central clinical laboratory test, offering unmatched sensitivity and simplicity. Moreover, the nanoparticle-catalyzed microbubbles also bridge the “nano” world to the “micro” world in a brand new way.



MDA schematic chart

Quantitating ultra-low concentrations of protein biomarkers is critical for early disease diagnosis and treatment. However, most current point-of-care (POC) assays are limited in sensitivity to meet this clinical need. Herein, we introduce an ultra-sensitive and facile microbubbling digital assay readout method toward POC quantitation of protein biomarkers requiring only bright-field smartphone imaging. Picolitre-sized microwells together with platinum nanoparticle labels enable the discrete “visualization” of protein molecules via immobilized-microbubbling with smartphone. We also use computer vision and machine learning to develop an automated image analysis smartphone application to facilitate accurate and robust counting. Using this method, post-prostatectomy surveillance of prostate specific antigen (PSA) can be achieved with a detection limit of 2.1 fM (0.060 pg/mL), and early pregnancy detection using bhCG with a detection limit of 0.034 mIU/mL (2.84 pg/mL) .  The results are further validated using clinical serum samples against clinical and research assays. This work provides the proof-of-principle of the microbubbling digital readout as an ultra-sensitive technology with minimal requirement for power and accessories, facilitating future POC applications.

MDA PSA result



Lateral Flow Assay

Lateral Flow Assay Ruler

Lateral flow assay (LFA) is a well-established platform for point-of-care (POC) testing due to its low cost and user friendliness. Conventional LFAs provide qualitative or semi-quantitative results, and require dedicated instruments for quantitative detection. Here we developed an “LFA Ruler” for quantitative and rapid readout of LFA results, using a 3D printed strip cassette and a simple, inexpensive microfluidic chip. Platinum nanoparticles are used as signal amplification reporters, which catalyze the generation of oxygen to push ink advancement in the microfluidic channel. The concentration of target is linearly correlated with the ink advancement distance. The entire assay can be completed within 30 minutes without external instrument and complicated operations. We demonstrated quantitative prostate specific antigen testing using LFA ruler, with a limit of detection of 0.54 ng/mL, linear range 0-12 ng/mL, and high correlation with clinical gold standard assay. The LFA ruler achieves low cost, quantitative, sensitive and rapid detection, which has great potential in POC testing and can be extended to quantify other disease biomarkers.


Lateral Flow Assay Ruler


Rapid Fentanyl Screening LFA Strip

Rapid identification of fentanyl at the point of care (POC) is critical. Urine fentanyl concentrations in overdose cases start at single-digit ng/mL. No fentanyl POC assay with cutoff at single-digit ng/mL is available. A competitive lateral flow assay (LFA) was developed using gold nanoparticles and optimized for rapid screening of fentanyl in 10 minutes. Urine samples from two cohorts of Emergency Department (ED) patients were tested using the LFA and a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The two cohorts consisted of 218 consecutive ED patients with urine drug-of-abuse screen orders, and 7 ED patients with clinically suspected fentanyl overdose, respectively. The LFA detected fentanyl (≥1 ng/mL) and major metabolite norfentanyl (≥10 ng/mL) with high precision. The assay demonstrated no cross-reactivity with amphetamine, cocaine, morphine, tetrahydrocannabinol, methadone, buprenorphine, naloxone and acetaminophen at 1000 ng/mL, and had 0.03%, 0.4% and 0.05% cross-reactivity with carfentanil, risperidone and 9-hydroxyrisperidone, respectively. In 218 consecutive ED patients, the prevalence of cases with fentanyl ≥1 ng/mL or norfentanyl ≥10 ng/mL was 5.5%. The clinical sensitivity and specificity (95% confidence interval (CI)) of the LFA were 100% (75.8-100%) and 99.5% (97.3-99.9%), respectively. The positive and negative predictive values (95% CI) were 92.3% (66.7-98.6%) and 100% (98.2-100%), respectively. The concordance between the LFA and LC-MS/MS was 100% in the 7 suspected fentanyl overdose cases (5 positive, 2 negative). The LFA is able to detect fentanyl and norfentanyl with high clinical sensitivity and specificity in the ED population with rapid fentanyl screening needs.



Fentanyl Screening Strip chart