HIGHLY SENSITIVE MICROFLUIDIC SENSOR ARCHITECTURE FOR SERS MEASUREMENTS OF HORMONES

Today, there is considerable interest in the development of microfluidic chips that can be integrated into various analytical devices and further used for chemical or biological research. The microfluidic approach can reduce the size of all types of analytical laboratory equipment’s and improve their technological performances. In addition, in combination with Surface Enhanced Raman Scattering (SERS), microfluidic devices can detect unprecedentedly low concentrations of targeted analytes. SERS provides a significant amplification of the nonlinear optical response of a molecule adsorbed on a suitable plasmon-active (most often silver or gold) surface (for example nanograting, nanoparticles, etc.). On the other hand, microfluidics allows a substantial reduction in the volume of sample required for analysis. In this work, an approach based on a combination of SERS and microfluidics was implemented and enables the detection of low concentrations of the target analyte hormones. Proposed preparation of microfluidic chip comprises several subsequent steps: (i) preparation of microfluidic platform using 3D printing; (ii) laser assisted creation of patterned surface, further covered by a thin Au layer, ensuring the excitation of surface plasmon polariton in the microfluidic canal, (iii) subsequent grafting of -NH2 groups, and their activation for hormones entrapping. The rapid, reproducible, and ultrasensitive detection of hormones Bisphenol, Estradiol, Estriol (< 10 M) was achieved.


INTRODUCTION
Sensors that determine the concentration of hormones, especially estrogens, are of particular interest for medical and environmental research [1]. Even a small, uncontrolled use of hormones by a person can cause adverse health effects, since certain types of hormones have been shown to cause cancer, heart disease and stroke [2]. Thus, there is a need for the determination of hormones to control their concentration in water and other biological fluids [3,4].
Today, there are several approaches to detecting hormones such as liquid chromatography and mass spectrometry [5,6]. However, they are quite complex, expensive, and time-consuming to analyse and results interpretation. Also, in recent years, sensors operating on the principle of surface plasmon resonance have become popular [7], but they have low selectivity and detection limit, which restricts their practical utilization. Therefore, the development of a plasmon-based (bio)sensor that will make it possible to carry out analysis quickly and reliably even with a small sample volume remains unsolved task.
The microfluidic approach makes it possible to reduce the size of all types of analytical laboratory equipment and improve their technological properties [8,9]. In addition, when combined with surface enhanced Raman scattering (SERS), microfluidic devices can detect unprecedented low concentrations of target analytes even with a small sample volume [10][11][12][13]. SERS provides a significant enhancement of the nonlinear optical response of a molecule adsorbed on a suitable metal (most often silver or gold) surface, while microfluidics can significantly reduce the sample requirements for analysis. In this work, we propose an approach based on a combination of SERS and microfluidics, which made it possible to identify low concentrations of the target analytes -hormones.

Microfluidic chip preparation
The microfluidic chip was made using 3D printing. The model for printing was designed in the program COMSOL Multiphysics. After fabrication, the microfluidic micromixer was washed with isopropyl, dried at 40° C for 24 h and additionally irradiated by UV-source for 30 min.
The Au thin films were deposited onto the patterned surface by vacuum sputtering (thickness 30 nm).

Diazonium modification and diazotization
Au grating was electrochemically modified in 3 mM fresh water solutions of ADT-NH2 without addition of any electrolytes, under the potential −5 V for 6 min. After modification, chips were rinsed under sonication sequentially with deionized water, ethanol, and acetone for 15 min, and dried in desiccator for 3 h.
The sample was immersed in a 15 ml methanol solution of p-Toluenesulfonic acid monohydrate and 0.3 ml of tert-Butyl nitrite was added. After activation chips were rinsed under sonication with MeOH for 15 min and dried in desiccator for 3 h.

Raman Spectroscopy Investigations-Concentration Dependence
For microfluidic SERS measurements, the chips were pumped with water solutions of Bisphenol, Estradiol and Estriol. Raman spectra were collected on portable ProRaman-L spectrometer (Laser power 35 mW) with 785 nm excitation wavelengths. Spectra were measured in the 2000-400 cm -1 wavenumber range with 60 s accumulation time and averaged 3 times.

RESULTS AND DISCUSSION
The preparation of the proposed SERS microfluidic chips consists of several stages, as schematically shown in Figure 1. First, a 3D microchannel was made using 3D printing technology and covered with a thin layer of Su-8. In the second stage, the periodic structure was created on the Su-8 by irradiation with excimer laser and covered with a thin layer of gold. Further the chip surface was grafted with -NH2 groups and activated to obtain a N=N + reactive sites, that allows hormones capturing. In the next step, we used two natural steroidal estrogens (estradiol and estriol) and one non-steroidal estrogen (bisphenol A) as model analytes for the study. We compare the SERS spectra, measured on SERS active substrate after hormones capturing in microfluidic regime with Raman spectra of targeted analyte in bulk. In all cases, the perfect coincidence of peak position and relative intensities (after spectra subtraction) was observed, indicating the success of proposed experimental route. In turn, Figures 2B-D show the calibration curves, prepared with more intensive, characteristic hormones SERS band. From calibration curves it is evident, that all used hormones were successfully entrapped detected up to 10 -8 M concentration. Comparison of obtained measurements uncertainty with typical errors from Figures 2B-D allows us to claim that by the proposed method it is possible to determine investigated hormones concentration with high precision.

CONCLUSION
In this work, we present the novel design of microfluidic-based chip for SERS detection of Bisphenol A, β-Estradiol and Estriol. The microfluidic chip was designed in Comsol and fabricated using 3D printing. The proposed detection approach is simple, fast, sensitive, and versatile, with great potential in hormone detection applications to monitor the safety of water and environment.