Electrospun biotin- and streptavidin-coated quartz crystal microbalance surfaces: characterization and mass sensing performance using OpenQCM

doi:10.21203/rs.3.rs-2995721/v1

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Electrospun biotin- and streptavidin-coated quartz crystal microbalance surfaces: characterization and mass sensing performance using OpenQCM

https://doi.org/10.21203/rs.3.rs-2995721/v1

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In this study, a quartz crystal microbalance (QCM) sensor surface was coated with biotin and/or streptavidin using the electrospinning method. The coated surfaces were analyzed using the Raman spectroscopy method. QCM measurements were carried out using the OpenQCM platform. The results indicate that the electrospinning method can be used to coat QCM surfaces with biotin and/or streptavidin and that the coated surfaces exhibit distinct morphological and spectroscopic properties. The QCM measurements showed that the coated surfaces are highly sensitive to changes in mass, indicating their potential for use in biosensing applications. Overall, this study provides new insights into the use of QCM sensors coated with biotin and/or streptavidin for biological sensing and detection applications.

Atomic and Molecular Physics

Analytical Chemistry

Structural Biology

Quartz crystal microbalance (QCM)

Biotin

Streptavidin

Electrospinning

Surface coating

Raman spectroscopy

Biosensing

Streptavidin, a protein capable of binding to biotin, exhibits a homotetrameric structure consisting of 126 amino acid residues in each identical monomer. Its secondary structure is characterized by the formation of an anti-parallel eight-stranded β-barrel tertiary structure [1]. The Trp120 residue in each monomer is responsible for connecting the monomer to its adjacent subunits, resulting in the formation of functional dimers within the tetramer. While the precise function of streptavidin remains unclear, previous research suggests its involvement in microbial defense mechanisms. The strong non-covalent bond between biotin and streptavidin has attracted significant attention in various scientific fields, owing to its exceptional specificity and high affinity, estimated to be in the femtomolar range (K_d = 10^− 13 − 10^− 15 M). This affinity is approximately an order of magnitude greater than that observed in well-known antigen-antibody interactions. The unique binding affinity displayed by the streptavidin-biotin complex possesses remarkable characteristics. Regions of the protein involved in interactions with other proteins or targets typically exhibit high flexibility and plasticity. Moreover, these regions tend to be located on the exposed surface of the protein. An essential segment of streptavidin, known as the apo-state streptavidin loop (L3/4; residues 45–52), plays a crucial role in biotin interaction and governs the binding process [2]. Streptavidin, biotin and Streptavidin-Biotin complex structures are presented in Fig. 1. Quartz crystal microbalance (QCM) sensors offer precise analysis of environmental changes, achieving high resolution at levels around ng/cm². They are particularly useful for accurately monitoring humidity [3], as well as detecting and analyzing atmospheric pollutants or organic chemicals [4].

QCM measurements were conducted utilizing a custom designed OpenQCM setup (Fig. 2., lower panel). Quartz crystal frequency measurements were performed using a PC. The measurements were carried out at the fundamental frequency, and a simultaneous collection of dissipation data was accomplished. Data acquisition was facilitated through the utilization of open-source OpenQCM software and Teensy firmware. The lab-made system was augmented with the integration of the Teensy microcontroller as the hardware component. Figure 2 illustrates phase and amplitude data obtained from the fundamental mode of a 10 MHz QCM (Quartz Crystal Microbalance) driven in an open-loop configuration. The quartz crystals utilized in the experiments were 5 mm in size (Raltron 18pf-HC49U) and incorporated electronic circuit elements with silver electrodes of a specific diameter. Q-values were measured at a temperature of approximately 20K employing the available system, yielding resonance frequencies of 9996348.043 Hz (stdev = 54.319).

Samples of biotin at concentrations of 5 mg/mL, 0.5 mg/mL, and 0.05 mg/mL were prepared, along with streptavidin samples at concentrations of 1.5 mg/mL, 0.5 mg/mL, and 0.05 mg/mL. The prepared biotin and streptavidin samples were loaded into a 500 µL Hamilton syringe (Model No. 80830; Hamilton Company, NV). Fluid flow was controlled using a syringe pump (Harvard PHD ULTRA, Syringe Pump Infuse/Withdraw Programmable), while a positive high voltage (SRS PS350, Sunnyvale, CA) was applied to the syringe tip. The QCM sensor, acting as a collector, was grounded to initiate the electrospraying process.

We have conducted an investigation into the correlation between the distance from the syringe tip to the collector (measured using a QCM sensor) and the electrospraying current. The purpose was to ascertain the impact of distance on the stability of the electrospraying process, which exhibited an inverse proportional relationship (Fig. 3.).

Biotin samples were meticulously dispensed using a flow rate of 2 µL/min, employing an accelerating potential of 5 kV, and maintaining a syringe tip-to-ground electrode distance of 5–8 cm. The samples were subjected to QCM measurements, which were carried out for a duration of 10 seconds (Fig. 4.). The experimental results clearly demonstrate that the deposition of biotin samples induces a frequency decrease of approximately 10 Hz on the resonance frequency. Remarkably, the anticipated frequency pattern identified in the biotin samples did not manifest in the case of streptavidin. Rather, a substantial decline in frequency within the 150 to 200 Hz range was recorded, alongside an anomalous value in one sample and a frequency reduction of approximately 10 Hz in another sample, diverging from the results of the remaining three measurements. These inconsistencies may potentially arise from experimental inaccuracies. Nevertheless, when the process was conducted utilizing an equivalent sample flow rate, a more pronounced decrease in frequency was observed, which can be attributed to the molecular weight of streptavidin.

Additionally, we have performed Raman microscopy measurements to evaluate the surfaces of the QCM sensors coated with biosamples and gain a deeper understanding of the observed variations. We utilized a 532 nm laser excitation with a power of 0.56 mW and a Leica 20× objective to capture Raman spectra of the QCM sensors. The spectra were acquired from two sides: the (black) quartz side and the (red) silver-coated side, as shown in Fig. 5. Furthermore, we presented Raman spectra of biotin-coated QCM sensors (with black representing the pure biotin sample) in Fig. 6, and streptavidin-coated QCM sensors (with black representing the quartz part) in Fig. 7.

ACKNOWLEDGEMENT

This work was supported by the Scientific and Technical Research Council of Turkey (TUBITAK) under Grant No. 118C476 and Grant No. 122F301. However, the entire responsibility of the publication belongs to the authors of the publication. The financial support received from TÜBİTAK does not mean that the content of the publication is approved in a scientific sense by TÜBİTAK. The authors acknowledge ME Research and Technologies (MERT Electronics) for providing the QCM sensors and OpenQCM hardware.

Hendrickson,W. A.,Pähler,A.,Smith,J. L.,Satow,Y.,Merritt,E. A.,&Phizackerley,R. P.(1989).Crystalstructureofcorestreptavidindeterminedfrommultiwavelengthanomalousdiffractionofsynchrotronradiation.ProceedingsoftheNationalAcademyofSciences,86(7),2190–2194.
Ayan,E.,Yuksel,B.,Destan,E.,Ertem,F. B.,Yildirim,G.,Eren,M.,…DeMirci,H.(2022).Cooperative allostery and structural dynamics of streptavidin at cryogenic and ambient temperature.Communications Biology,5(1),73.
Yu,X.,Chen,X.,Yu,X.,Chen,X.,&Ding,X.(2021).A quartz crystal microbalance (QCM) humidity sensor based on a pencil-drawn method with high quality factor.IEEE Transactions on Electron Devices,68(10),5149–5154.
Fauzi,F.,Rianjanu,A.,Santoso,I.,&Triyana,K.(2021).Gas and humidity sensing with quartz crystal microbalance (QCM) coated with graphene-based materials–A mini review.Sensors and Actuators A: Physical,330,112837.

Competing interests: The authors declare no competing interests.

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Electrospun biotin- and streptavidin-coated quartz crystal microbalance surfaces: characterization and mass sensing performance using OpenQCM

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Abstract

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I. INTRODUCTION

II. MATERIALS AND METHODS

III. RESULTS AND DISCUSSIONS

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ACKNOWLEDGEMENT

References

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