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. 2025 Jul 1;15(7):417.
doi: 10.3390/bios15070417.

Droplet-Based Measurements of DNA-Templated Nanoclusters-Towards Point-of-Care Applications

Jonas Kluitmann  1 , Stefano Di Fiore  2 , Greta Nölke  2 , Klaus Stefan Drese  1
Affiliations

Droplet-Based Measurements of DNA-Templated Nanoclusters-Towards Point-of-Care Applications

Jonas Kluitmann et al. Biosensors (Basel). .

Abstract

In this work, we investigate the fundamental usability of fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) as sensors for Point-of Care-Testing (PoCT) applications. We developed a microfluidic platform for the generation of droplets containing DNA-AgNCs in defined, different chemical environments. The droplets are read out fluorescently at two different emission wavelengths. For the pre-evaluation for the usage of biologically relevant matrices with DNA-AgNCs, the response of two different DNA-AgNCs to a variation in pH and sodium chloride concentration was acquired. Our compact and simple setup can detect DNA-AgNCs well below 100 nM and allows the characterization of the fluorescence response of DNA-based biohybrid nanosensors to changes in the chemical environment within short measurement times. The model DNA-AgNCs remain fluorescent throughout the physiologically relevant chloride concentrations and up to 150 mM. Upon shifts in pH, the DNA-AgNCs showed a complex fluorescence intensity response. The model DNA-AgNCs differ strongly in their response characteristics to the applied changes in their environments. With our work, we show the feasibility of the use of DNA-AgNCs as sensors in a simple microfluidic setup that can be used as a building block for PoCT applications while highlighting challenges in their adaption for use with biologically relevant matrices.

Keywords: DNA-templated silver nanoclusters; Point-of-CareTesting; biohybrid nanosensors; fluorescence; microfluidics; nanoclusters; segmented flow.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematics of the fluorescence readout cell and image of the laboratory setup. (a) Exploded view of the fluorescence flow-through measurement cell, (b) top-view of the optical setup in the measurement cell, (c) picture of the laboratory setup without computer for data logging. 1: emission LED board; 2a: filter holder; 2b: bandpass filter; 3: measurement cell slip lid; 4a: SiPM holder body; 4b: SiPM holder lid; 5: collimating lens; 6: trench for the tubing; 7: measurement cell body; 8: SiPM bias generator and amplifier module; 9: measurement cell electronics mount; 10: SiPM cable feedthrough; 11: syringe pumps; 12: droplet generator; 13: laboratory power supplies; 14: containment for the measurement cell. The LED constant current driver board and microcontroller board are not shown for clarity.
Figure 2
Figure 2
Normalized fluorescence emission spectra of the DNA-AgNCs in 20 mM NH4OAc. Excitation wavelength λex = 270 nm. The shaded ranges show the transmission windows of the bandpass filters at ODs ≤ 3 used in the setup.
Figure 3
Figure 3
The raw data of the SiPM intensity signal for the dilution of the DNA-AgNC construct Ag28b-1 for channel BP668. The encircled numbers correspond to the condition number and match those in Figure S2 and Table S2.
Figure 4
Figure 4
The concentration-dependent emission signal intensity of the DNA-AgNCs and linear fits of the datasets. (a) The signal of channel BP565 and (b) signal of channel BP668 for a dilution range of 5 µM to 12.5 nM for Ag19b-1 and Ag28b-1, respectively.
Figure 5
Figure 5
Fluorescence intensity of (a) Ag19b-1 and (b) Ag28b-1 in response to different concentrations of sodium chloride. Each datapoint consists of 33 ± 3 droplets, each representing one measurement. Error bars indicate the standard deviation.
Figure 5
Figure 5
Fluorescence intensity of (a) Ag19b-1 and (b) Ag28b-1 in response to different concentrations of sodium chloride. Each datapoint consists of 33 ± 3 droplets, each representing one measurement. Error bars indicate the standard deviation.
Figure 6
Figure 6
Fluorescence intensity of (a) Ag19b-1 and (b) Ag28b-1 in response to different pH values. Each datapoint consists of 33 ± 3 droplets, each representing one measurement. Error bars indicate the standard deviation.
Figure 7
Figure 7
Proposed sensors for ratiometric pH measurements: (a) the use of Ag19b-1 in channels BP565 and BP668 and (b) the use of Ag19b-1 in channel BP565 and Ag28b-1 in channel BP668. The values of the dashed line are measured by the secondary y-axis of the chart as indicated by the arrow.
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