Advanced Biosensor and Microfluidic Technologies for Rapid Pathogen Diagnosis and Cancer Research

Rapid Pathogen Diagnosis

• Current Limitations:

  • Pathogen identification currently takes up to $72$ hours, often involving PCR-based methods, leading to delays in patient treatment.

• Novel Approach:

  • Significantly reduces diagnosis time to approximately $2$ hours.

  • Utilizes biosensors capable of identifying pathogens even at very low concentrations.

• Biosensor Mechanism:

  • Carbon Nanotube (CNT): Acts as a probe delivery system, facilitating the entry of probes into bacteria via electroporation.

  • RNA Probe: A single-stranded probe that is complementary to a specific bacterial gene RNA (e.g., $16S$ rRNA).

  • Fluorophore-Quencher System:

    • In the absence of target bacterial RNA, a shorter quencher strand is bound to the fluorophore on the probe. This binding causes fluorescence quenching (no signal) due to a relatively higher free energy of the quencher-probe complex.

    • When target bacterial RNA is present, the probe binds to the target RNA, which leads to the detachment of the fluorophore from the quencher, resulting in the release of a detectable fluorescent signal.

• Specificity and Detection Capability:

  • Successfully identifies E. coli (indicated by a red signal) and P. aeruginosa (indicated by a green signal) simultaneously within the same experiment.

  • Demonstrated effective detection even with very low bacterial counts present in the sample.

  • Experimental videos confirm the system functions with viable pathogens, as bacteria are observed moving.

High-Throughput Antibiotic Resistance Screening

• Microfluidic Platform:

  • Bacteria are introduced into individual microfluidic channels, with each channel containing a small, defined population (e.g., a "column" of bacteria).

• Growth Monitoring:

  • The system tracks bacterial growth over time, observing an increase in length or count (e.g., from one entity to ten entities) in the absence of antibiotic treatment.

• Resistance Assay Procedure:

  • Sample Preparation: Healthy human blood samples cultured with bacteria, as well as patient samples positive for E. coli, are loaded into the microchannels.

  • Incubation and Treatment: Samples are incubated for $30$ minutes, after which specific antibiotics are introduced.

  • Outcome Assessment:

    • If the bacteria are sensitive to the applied antibiotic, their growth ceases (e.g., only one original cell remains).

    • If the bacteria are resistant, their growth continues uninterrupted.

  • Time Efficiency: This method significantly reduces the time for antibiotic resistance determination to approximately $1$ hour, a dramatic improvement over traditional multi-day methods.

  • Example: Sample number $6$ was specifically identified as resistant to antibiotic C.

Pathogen Identification Based on Physical Properties

• Core Principle: Utilizes a microfluidic device to separate and identify bacterial pathogens based on their distinct size and shape characteristics.

• Separation Mechanism:

  • Bacteria are introduced into microfluidic channels, and varying pressures are applied.

  • Size-Based Trapping: Bacteria with larger sizes are easily trapped at lower fluid pressures. Conversely, smaller bacteria require higher pressures to be effectively trapped and separated.

• Shape-Based Classification:

  • Coccus: Identifies spherical-shaped bacteria.

  • Bacillus: Identifies rod-shaped bacteria.

  • These morphological classifications are performed by observing bacterial behavior under different applied pressures on the microscope.

• Clinical Application and Benefits:

  • Successfully applied to clinical samples. For instance, in a cohort of $25$ patients, $7$ patients were found to have pathogens resistant to