# Multiplex on-chip detection of Aspergillus by integrated ultrasonication-based bead beating lysis and magnetic beads direct amplification

## TL;DR

This paper introduces a fast and sensitive microfluidic chip system for detecting Aspergillus spores in clinical and environmental samples using ultrasonication and magnetic beads.

## Contribution

The study presents an integrated USBB-MBDA system for rapid, sensitive, and automated detection of multiple Aspergillus species.

## Key findings

- The system achieved 85.5% lysis efficiency for Aspergillus at 105 spores per sample.
- It enabled triplex detection of A. fumigatus, A. flavus, and A. niger with sensitivity as low as 10 spores per test.
- The workflow completed sample-in to result-out in 35 minutes with 100-fold improved detection sensitivity.

## Abstract

Introduction: Invasive aspergillosis (IA) is a life-threatening disease in immunocompromised individuals, creating an urgent need for rapid, sensitive, and user-friendly methods for early detection of Aspergillus spores in clinical and environmental samples.

Methods: In this study, we developed a rapid ultrasonication-based beads beating (USBB) method for cell lysis and fungal DNA release, along with an integrated magnetic beads-based direct amplification (MBDA) method utilizing microfluidic chip technology. Our flexible thin-film microfluidic chip was developed to enable immediate contacting with an ultrasonic oscillator for nucleic acid extraction.

Results and discussion: The chip achieved a lysis efficiency of 85.5% for Aspergillus at 105 spores per sample. Magnetic beads releasing Fe ions at 75-860 ng/mL allowed high-efficiency direct PCR amplification. Complete transfer of the nucleic acid extract into PCR amplification led to a 100-fold improvement in detection sensitivity. The integrated USBB-MBDA system completed the entire workflow from sample-in to result-out within 35 min (5 min fungal DNA release and 30 min TaqMan assay). Using this approach, we achieved simultaneous triplex detection of Aspergillus
fumigatus, Aspergillus flavus, and Aspergillus niger, with a sensitivity as low as 10 spores per test. Validation on 37 clinical samples showed complete concordance with MALDI-TOF mass spectrometry (MS). This study establishes an integrated nucleic acid extraction and magnetic bead–enabled direct amplification strategy, providing a versatile approach for the development of automated analytical platforms with broad applicability to in vitro diagnostics.

Detection of three invasive Aspergillus species on a microfluidic chip using ultrasonic beads beating for cell lysis combined with magnetic beads–based direct amplification.Workflow diagram showing preparation and detection of spore samples using a thin-film microfluidic chip with magnetic and glass beads, processed by an ultrasonic oscillator, followed by detection with portable qPCR and results readout, total time thirty-five minutes. A line chart visualizes fluorescence over cycles for A. flavus, A. niger, A. fumigatus, and a negative control.

Detection of three invasive Aspergillus species on a microfluidic chip using ultrasonic beads beating for cell lysis combined with magnetic beads–based direct amplification.