Breaking the barrier to biomolecule limit-of-detection via 3D printed multi-length-scale graphene-coated electrodes

Sensing of clinically relevant biomolecules such as neurotransmitters at low concentrations can enable an early detection and treatment of a range of diseases. Several nanostructures are being explored by researchers to detect biomolecules at sensitivities beyond the picomolar range. It is recognized, however, that nanostructuring of surfaces alone is not sufficient to enhance sensor sensitivities down to the femtomolar level. In this paper, we break this barrier/limit by introducing a sensing platform that uses a multi-length-scale electrode architecture consisting of 3D printed silver micropillars decorated with graphene nanoflakes and use it to demonstrate the detection of dopamine at a limit-of-detection of 500 attomoles. The graphene provides a high surface area at nanoscale, while micropillar array accelerates the interaction of diffusing analyte molecules with the electrode at low concentrations. The hierarchical electrode architecture introduced in this work opens the possibility of detecting biomolecules at ultralow concentrations.

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Life sciences
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Life sciences study design
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Sample size

Data exclusions
Replication Statement is included in the manuscript which is "All relevant data that support the findings of this study are presented in the manuscript and supplementary information file. Source data are available from the corresponding author upon reasonable request" We would like to clarify that this is an engineering study for a biosensing technique rather than a life sciences study. The choice of sample sizes is given below.
a) For the repeatability study of the sensor (Figure 8c), 30 biologically independent experiments (n=30) across 10 sensors evenly split between 2D and 3D configuration were performed. Use of 15 experiments across 5 devices (randomly chosen with respect to the manufacturing sequence) gives a good indication of normality of data from design of experiments (Science advances, 2020, 1, 6(32), eabc4250; and Advanced Materials, 2021, 33, 2006647). Figure 8a and 8b have the same sample size justification. Once repeatability was established, storage stability of the device was studied with 15 biologically independent experiments across 12 days at an interval of three days were performed on a sensor to obtain Figure (2):419-24). Randomization with respect to time was not applicable because data was collected in a time sequence. b) Sample size for spiked studies (Figure 6 and S10) was 60 biologically independent experiments (n=60) across two sensors evenly split between human plasma ( Figure 6b) and artificial serum ( Figure S10b). Two sensors were randomly chosen with respect to the manufacturing setup. One sensor was used to test 30 samples of spiked dopamine concentration (n=30; biological independent experiments) which were made in human plasma at five different concentration while other sensor was used to test 30 samples of spiked dopamine concentration (n=30; biological independent experiments) were made in artificial serum at five different concentration. Since the dopamine concentration was same across different dilution of human plasma and artificial serum, a total of 15 readings are obtained each with and without dopamine. This sample size is higher than 5 samples required to assess normality of the data. Figure 6a and 10Sa have the same sample size justification. c) Sample size for selectivity studies (Figure 7) was 66 biological independent experiments (n=66) across two sensors evenly split between 2D ( Figure 7b) and 3D sensor (Figure 7d) configuration. Two sensors were randomly chosen with respect to the manufacturing setup. The 2D sensor was used to test 33 samples that were made in different neurotransmitters with a fixed dopamine concentration (Figure 7b). Five different neurotransmitters were chosen in this experiment and a total of 33 experiments (15 experiments with other nuerotransmitters only and 18 experiments where a combination of other neurotransmitters and dopamine were used. This sample size was sufficient to establish the normality of the data and the selectivity of the device, which was the purpose of this study. The same argument holds for the 3D sensor configuration (sample size, n=33, Figure 7d). Figure 7a and 7c have the same sample size justification. d) The aim for the experiment described in Figure 5 was to establish in-vitro analysis of the sensor in presence of rabbit serum and fetal bovine serum as well as demonstrate that the 3D sensor has a higher signal in all the cases. Sample size for this study was 42 (n=42, biological independent experiments) across two sensors evenly split between 2D ( Figure 5b) and 3D (Figure 5d) configuration. Use of 21 experiments (n=21) across one 2D device (sensor chosen with respect to the manufacturing setup). A total of seven samples (21 readings) gives a good indication of normality of data from design of experiments (Figure 5b). Similarly, a same sample size (n=21) was used for 3D sensor configuration (Figure 5d). The two sensors used to collect data in Figure 5b and 5d were chosen randomly with respect to the manufacturing setup. The time sequence of experiments is as appears in the x-axis of the figures. In confirm that the experiments are not biased, pbs (i.e., baseline) was tested at the end of each sequence to confirm hat the signal goes back to the baseline. Figure 5a and 5c have the same sample size justification. e) The aim of the titrate experiment described in Figure 4 was to carry out the titrate analysis and establish that the current is high for the 3D sensor compared to the 2D sensor. The sample size for this analysis was 63 biologically independent experiments (n=63) across three sensor configurations such as 2D (0×0), 3D (4×4) and 3D (10×10). For a 3D (10×10) sensor, a total sample size of eight (total readings from 24 biological independent experiments; n=24) was chosen for the serial titrate measurements (Figure 4f). Each concentration of dopamine was assessed with three repeat measurements (Nat. Comm.,12, Article number: 4039, 2021). The same sample size was used for the other two configurations as shown in Figures 4b and 4d. Since this is a titrate-type dose-dependent experiment, the dopamine concentration was increased serially (Biosens. Bioelectronic. 138, 2019, 111310). Figure 4a, 4b and 4c have the same sample size justification.
No data was excluded from the analysis For each experiments, we have repeated three replicate measurements as stated above Section of Sample Size.