SERS-based ultrafast and sensitive detection of luteinizing hormone in human serum using a passive microchip

Accepted Manuscript Title: SERS BASED ULTRAFAST AND SENSITIVE DETECTION OF LUTEINIZING HORMONE IN HUMAN SERUM USING A PASSIVE CHANNEL MICROCHIP Authors: Belma Gjergjizi, Ferah C ¸ o˘gun, Ender Yıldırım, Merve Eryılmaz, Yes¸im Selbes, Necdet Sa˘glam, U˘gur Tamer PII: DOI: Reference:

S0925-4005(18)30895-5 https://doi.org/10.1016/j.snb.2018.05.001 SNB 24656

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

20-11-2017 25-4-2018 1-5-2018

Please cite this article as: Belma Gjergjizi, Ferah C ¸ o˘gun, Ender Yıldırım, Merve Eryılmaz, Yes¸im Selbes, Necdet Sa˘glam, U˘gur Tamer, SERS BASED ULTRAFAST AND SENSITIVE DETECTION OF LUTEINIZING HORMONE IN HUMAN SERUM USING A PASSIVE CHANNEL MICROCHIP, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SERS BASED ULTRAFAST AND SENSITIVE DETECTION OF LUTEINIZING HORMONE IN HUMAN SERUM USING A PASSIVE CHANNEL MICROCHIP

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Belma Gjergjizia, Ferah Çoğunb, Ender Yıldırımb, Merve Eryılmazc, Yeşim Selbesc, Necdet Sağlama, Uğur Tamerc, *

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Hacettepe University, Faculty of Science, Department of Biology, 06800, Beytepe, Ankara, Turkey

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Cankaya University, Department of Mechanical Engineering, 06790, Etimesgut, Ankara, Turkey

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Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry, 06330, Etiler, Ankara, Turkey

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* Corresponding Author at Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry, 06330, Etiler, Ankara, Turkey. E-mail address: [email protected]

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Graphical abstract

Highlights

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Antibody modified gold nanoparticles enable the capture human luteinizing hormone (LH).. The gold nanoparticles are also modified as a Raman reporter. Sandwich immunoassay for detection of the analyte. A passive channel microchip is designed for fast analysis of LH with small amount of reagents. Surface-enhanced Raman spectroscopy lowers the limit of detection. Successful recovery of LH spiked in serum samples via the microchip.

ABSTRACT

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Human luteinizing hormone (LH) is an important analyte for doping control analysis since it increases the athletic performance. However, traditional methods to detect LH have few disadvantages, such as long analysis duration, waste disposal problem and sample matrix effect. Addressing these problems, surfaceenhanced Raman spectroscopy based LH analysis using a passive microfluidic chip was developed and optimized. Antibody modified magnetic gold nanoparticles captured the LH and then, 4-aminothiophenol (4-ATP) labeled nanoparticles formed the sandwich immunoassay structure. The complex and the other reactions occurred in different chambers of the chip. The SERS signals of 4-ATP were recorded from the chamber and the system was shown to detect 0.036 IU L-1 in serum samples. The performance of the immunoassay was compared to all other methods and the proposed assay was the fastest analysis of LH without any problems associated with the sensitivity. The shorter analysis time was recorded because of the chip enables the control of all reactions in one place and there was no requirement of a specialized laboratory.

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Keywords: Luteinizing hormone, gold nanoparticle, SERS, microchip, immunoassay.

INTRODUCTION

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The luteinizing hormone (LH) is a glycoprotein hormone synthesized and secreted by the gonadotropic cells in the anterior pituitary gland. It consists of α and β subunits. TSH, FSH, CG share common 92 amino acid α subunits, while the human gene is located on the 6q12-q21 chromosome [1]. The World Anti-Doping Agency (WADA) put LH on the list of prohibited items for male athletes [2]. This hormone allows testes to convert cholesterol to testosterone in Leydig cells [3], which increases the number of red blood cells in the bloodstream. Since blood cells carry oxygen up to the muscles from the lungs, a higher concentration in the blood increases the aerobic capacity (VO2 max) and endurance of an athlete, thereby increasing the athletic performance [4]. In hospitals and laboratories, conventional methods such as enzyme-linked immunosorbent (ELISA), homogeneous and heterogeneous immunosorbent assays are performed for the identification of biological markers present in body fluids. However, these methods are time-consuming and use copious amounts of solvent that must eventually be disposed of. In addition, the requirement of different sample preparation steps and expensive reagents are other drawbacks of the methods [5]. For the detection of LH, ELISA and other immunoassay methods are performed. The traditional methods of LH in human fluids are ELISA [6-

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10] and radioimmunoassay (RI) [11, 12]. High-performance liquid chromatography is also a conventional method in pharmaceutical analysis and clinical diagnosis [13, 14] while its application in an immunoassay is not as good as in capillary electrophoresis-based analysis. In addition, there was no report for detection of LH using HPLC. Even though these methods are reliable and accurate, there are problems associated with low sensitivity and matrix effects. The use of isotopes in IR is the basic problem which has not been totally resolved [15]. The chemiluminescence based immunoassay for LH detection has comparable detection limits however the cross-reactivity and the adoption of CL enhancer are the problems they still suffer [16]. Gas chromatography-mass spectroscopy and fully automated systems for detection of LH were also reported [17].

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There have been many studies on biosensors in recent years to establish an ideal analysis platform for body fluids. Biosensors are analytical devices that can selectively determine the analyte in a mixture. Generally, antibodies are used as a molecular recognition agent. The complex formed between the antibody and antigen shows the high complexity and sensitivity [18]. Immunomagnetic separation with the use of magnetic nanoparticles has been used in many studies since this separation enables successful isolation of the analyte [19]. In this study, a sandwich type biosensor was developed for LH detection by surfaceenhanced Raman spectroscopy (SERS). Among numerous protein detection methods, SERS has become an area of intense research as a highly sensitive probe for trace level detection of biomolecules such as proteins, bacteria [20] and DNA/RNA [21]. It can even be used in single molecule detection [22]. SERS technique may provide up to 102–1014 enhancement in Raman signal intensity [23], and this is sufficient to detect pico- to femtomolar amounts of biomolecules. In addition, it can be a powerful alternative for detection of low abundance protein because of its high sensitivity [24].

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Surface roughness is essential for producing uniform and high-intensity spectra in SERS measurement [25]. The enhancement shows a great promise in different sensor designs due to signal amplification of nanoparticles, which enables sensitive and selective target analysis [26,27]. There are many different analytes used in sensors from HIV-1 DNA to trypsin molecule or thyroid-stimulating hormone TSH [2830]. A paper based lateral flow immunoassay is one of the popular technique in point-of-care diagnostics with some limitation in its quantification analysis [31]. In the analysis bio analytes, the use of nanoparticles enables a quantitative analysis in the low concentration range. The most preferred enhancer is gold nanoparticles (AuNPs) regarding the easy preparation and modification, better biocompatibility and homogenous aggregation during the SERS effect [32] DNA of HIV-1 was successfully quantified with Raman reporter modified AuNPs and it was shown that nanoparticles enabled more sensitive detection compared to fluorescent or colorimetric methods [28]. Similarly, (TSH) was monitored lower than 0.5 IU/mL using the SERS nano-tag functionalized gold nanoparticles [30]. Silver nanoparticles (AgNPs) are also recommended as enhancer agent in SERS measurement. Chen et al. detected trypsin with 4mercaptopyridine (4-MPY)-modified AgNPs by anti-aggregation mechanism between protamine and trypsin. Besides controlling distance between substrate and the Raman reporter, in this work SERS assay was performed based on aggregation [29]. Therefore, it was clear that advantages of significant properties of nanoparticles enable excellent analytical performance. Herein, we also use gold nanoparticles for more sensitive detection and AuNPs were modified with 4Aminothiophenol (4-ATP) to monitor SERS signal. After the interaction of magnetic nanoparticle and LH, the signal was obtained from sandwich immunoassay. Different from paper-based devices or conventional batch technique, isolation of target protein and all other interactions with modified nanoparticles were

assembled in a microfluidic chip. In this way, a sensitive and selective analysis of LH was performed with a small requirement of the sample and other reagents. Moreover, the most significant improvement in the proposed system was the shorter duration of the analysis.

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In addition, there are different SERS reporters to monitor mechanism of the assay with characteristics Raman peaks [33]. The nanoparticles are modified with these reporters due to their many advantages in SERS measurement. First of all, the reporters are robust to laser irradiation because of the ultra-fast energy transfer from the excited molecules to metal surface [34]. Secondly, they have large signal intensity due to large SERS cross-sections, which values are higher by 102 times than those dye molecules or quantum dots [34]. Blinking in the system composed of silver nanoparticles and the analyte shows strong chemical effect rather than the electromagnetic (EM) mechanism. The blinking is correlated with the photoexcitation/emission and the plasmon resonance [35]. Thus, the main reason for it is a-photo-induced effect, where it is considerably suppressed by fast energy transfer. The spectral changes in SERS are induced by EM mechanism and chemical effects. The chemical effects are divided into three types: the resonance Raman effect, adsorption effect and charge-transfer (CT) effect [36]. These mechanisms are not independent and interact with each other. Especially, large SERS cross-section is induced by CT and resonance effect due to nanoparticle-molecule charge transitions [34-36].

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Nanoparticle immobilizing in a microchip is divided into four groups according to working principles: polymer entrapment, surface adsorption, covalent binding and magnetic attraction [37]. Nanoparticle immobilizing on an adhesive tape was tested as microchip substrate with detection of RG6 using SERS [37]. However, it is easy to perform and enables the reuse of microchip; the real sample sensing of the system is still a challenge. Most preferred microfluidic chips are continuous flow type, in which the samples and reagents generally flow through separate channels and mixed by using a micromixer. These systems often require a pump to provide the flow. Besides, it is often necessary to mount port connections to the chip inlets and outlets to transmit the working fluid from tubes or vial to the chip through small diameter tubing. Due to the use of off-chip components, the reliability of such devices is low and their use is relatively difficult. In this study, as an alternative to continuous flow microchip, a passive microfluidic chip was designed, in which the working fluid is transferred into the chip by using a pipette and kept stationary in micro chambers in the chip. The microfluidic chip consists of four chambers connected to each other by capillary valves [38]. The working fluids are delivered through the pipettes in each chamber respectively. The fluid fills the chamber by capillary action. When the capillary interface reaches the downstream end of the valve channel opening to the next chamber, the liquid cannot go further because of the meniscus pinning effect caused by the capillary pressure at the interface opposing the flow. In addition, without any complex process and the need of any reagents, nanoparticles can be moved with a magnet in this passive microchip.

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Compared with traditional methods, the proposed method offers a few advantages. The main advantage is that passive microchip-based immunoassay has high sensitivity and there are no sample matrix effects compared to ELISA. In addition, the time for this method is faster because the immunoassay reactions occur in chambers, which allows rapid reaction in lesser amounts of solvents; and it consumes fewer reagents and requires fewer samples. The microfluidic chip has the novelty in transferring solutions to the chambers through the access holes by pipetting. The liquid must not flow through the adjacent chamber, thus the next chamber could be filled with another liquid without mixing. Therefore, the steps of immunoassay are controlled respectively, and only the target analyte was in the last chamber for measurement. In addition, the passive microchip is not designed for a specific analyte, it has the advantage of detecting a different type of analyte and it is compatible with any other nanoparticle-based immunoassay design. In this work,

the application of magnetic nanoparticles in passive channel microchips improved the speed and sensitivity of LH analysis in doping control analysis. The reaction parameters were optimized and discussed in detail, and the serum samples were used as a real sample by the proposed method.

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2. EXPERIMENTAL 2.1. Reagents and materials

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Luteinizing hormone, (LH Spec kit, and catalog code B031-101) standards and monoclonal mouse anti-β human luteinizing hormone IgG class antibodies (Anti-LH-Ab) were obtained from WallacOy (Mustionkatu 6, FI-20750 Turku, Finland). Synthetic control serum samples were obtained from Bio-Rad Laboratories (Hercules, California, USA). Iron (III) chloride (FeCl3) is obtained from FLUKA (Bucharest, Romania). Iron (II) sulphatehepta hydrate (FeSO4.7H2O), disodium phosphate (Na2HPO4), sodium hydroxide (NaOH), hydrogen tetrachloro Gold (III) (HAuCl 4), hydroxylamine hydrochloride, L-Ascorbic acid (C6H8O6), 99.9% Ethanol, Hexadecyltrimethylammonium bromide (CTAB), 11-Mercaptoundecanoic acid (11-MUA), 98% N-(3-Dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC), 98% NHydroxysulfosuccinimide sodium salt, potassium chloride (KCl), 2-(N-morpholino) ethanesulfonic acid (MES monohydrate), ethylenediaminetetraacetic acid disodium salt (EDTA), sodium phosphate (Na2HPO4) and potassium phosphate (KH2PO4) were obtained from Sigma–Aldrich (Taufkirchen, Germany and Saint Louis, USA). Silver nitrate (AgNO3), 4-aminothiophenol (4-ATP), ethanolamine (98%), sodium borohydride (NaBH4) and perchloric acid (HClO4) were obtained from MERCK (Darmstadt, Germany). Benzalkonium chloride (BCC) (50%) was obtained from TEKKIM. 2.2. Instrumentation

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Raman spectroscopy (DeltaNu Examiner Inc. Laramie, WY, U.S.A & Qimagin MicroPublisher-RTV-5.0, Canada) a Raman microscope with a 785-nm laser source, the moving coverslip microscope holder for the sample and the cooled charge-coupled device (CCD, at 0°C) detector was used for the measurement. The microscope objective has very important two functions: scanning the laser and collecting the Raman signal generated from the sample. For this reason, a good alignment of the objective is a must to achieve a high transmittance of the signal. SERS signals from 4-ATP were measured by focusing the laser beam on the hole of the last chamber of the microchip. The device parameters are set to 45 second data collection time, 10X objective, 30 μm laser spot area, 150 mW laser power and baseline correction was performed for all measurements. Transmission Electron Microscope (TEM) (JEOL, Jeol Ltd., Tokyo Japan) was used for characterization studies of nanoparticles. 2.3. Synthesis of gold magnetic nanoparticles

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To synthesis gold coated magnetic nanoparticles (Fe3O4@Au), magnetite nanoparticles were prepared by precipitation of Fe (II) and Fe (III) firstly. For this purpose, 5 mL of 1.28 M FeCl3 and 5 mL of 0.64 M FeSO4.7H2O were dissolved in deionized water and yellow-green color was observed in room temperature. Then, the solution was stirred vigorously until the iron salts were dissolved. Subsequently, 125 mL of 1 M NaOH was added dropwise to the mixture with stirring for 40 minutes and in the end precipitated Fe nanoparticles was black. Gold coating procedure was carried out in a sonicator to encapsulate the iron NPs with gold shells. For this purpose, 10 mg of Fe particles were suspended in 0.27 M EDTA which was prepared in 1 M NaOH. This solution was sonicated for 5 min. The particles were then centrifuged for 10

min at 10,000 rpm and the supernatant was discarded. The nanoparticles were washed with deionized water and the washing procedure was repeated three times. The precipitate was mixed with 7 mL of 0.1 M CTAB, 3 mL of 0.01 M HAuCl4, and 300 μL of 1 M NaOH. Then, the solution was stirred vigorously and 150 mg hydroxylamine was added to the mixture and stirred for extra 3 min. In the end, the color of the nanoparticle solution was dark red [39]. 2.4. Modification of magnetic nanoparticles for the immunoassay

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Modification of magnetic NPs was performed prior to SAM formation of molecules on the surface of Fe3O4@Au. For this purpose, 20 mM of 11-MUA solution was prepared in absolute ethanol. The magnetic NPs were mixed with this solution and it was kept in the shaker for 24 hours to form carbonyl groups on the gold surface. The excess of 11-MUA was removed with a washing step with MES buffer (pH 6.5). To activate the free carbonyl groups formed on the gold surfaces, 0.2 M EDC and 0.05 M NHS solutions were prepared in the same buffer solution. The NPs were mixed with these solutions for 45 minutes. Covalent bonding with amine groups resulted in the formation of amine-reactive NHS esters on the surface of the NPs. Immobilization of antibodies occurs by submersing Fe3O4@Au-MUA conjugate overnight in TrisHCl buffer (pH 7.8), BSA solution and 25 μgmL−1 antibodies solution. The antibody-modified NPs were washed two times with MES buffer. To avoid non-specific interactions, solution of ethanolamine (10 %, v/v) and D-alanine (4 mg mL−1) was prepared and the nanoparticles were stirred for an hour. The preparation of Fe3O4@AuNP-MUA-Ab conjugate was performed in two steps. Firstly, isolation of LH was achieved by utilizing antigen-antibody interaction. To make an efficient immune complex formation, LH hormone and Fe3O4@Au NPs were incubated for 45 minutes. Removal of unbound LH at the end of the interaction was completed by washing two times with 0.05 M MES buffer. In the end, the modification of Fe3O4@Au with LH was achieved and the NPs were transferred to the microchip for the formation of a homogeneous sandwich immunoassay.

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2.5. Synthesis and characterization of gold nanoparticles

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Rod shaped and spherical gold nanoparticles (AuNPs) were synthesized in this study. As a first step for the synthesis of rod-shaped gold nanoparticles, 7.5 mL of 0.1 M CTAB and 250 μL of 0.01 M HAuCl 4 were mixed and then 0.01 M NaBH4 solution was added to form seed solution. The color of the seed solution was pale orange at 30°C for 45 minutes. Secondly, 4.75 mL of 0.1 M CTAB, 100 μL of HAuCl4 and 60 μL of AgNO3 were mixed in a vial to obtain a dark orange color. After adding 250 μL of 0.01 M AA to the solution, the color was disappeared because of the gold reduction. Then, 10 μL of the seed solution was added and left for 3 hours and in the end dark blue color was the confirmation of the formation of gold nanorod particles. For the synthesis of spherical shaped gold NPs, same steps were followed however 0.1 M of BCC was used instead of 0.1 M of CTAB. At the end of the synthesis, the nanoparticles were dark red [40] and the characterization of AuNPs was performed with Transmission Electron Microscopy (TEM) and UV-visible spectroscopy. The size and shape information of nanoparticles are given in our previous work [39-40] and the same type of nanoparticles were used in the proposed method. 2.6. Modification of AuNPs with 4-aminothiophenol The synthesized spherical and rod-shaped AuNPs interacted with 50 mM of 4-ATP (Raman active molecule) for Raman labeling of nanoparticles. For this purpose, the AuNPs were mixed with this solution

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and was kept in the shaker overnight to form carbonyl groups on the gold surface. The excess of 4-ATP was removed by washing the particles with MES (pH 6.5) buffer. The SAMs of chemisorbed 4aminothiophenol has been used as the base interface for the deposition of the antibodies onto Au surface. The thiol group of 4-aminothiophenol binds to Au surface leaving the amino group free to bind with antibodies. It is believed that the 4-ATP SAM layer was first converted to a 4′-mercapto-N-phenylquinone diimine (NPQD) layer followed by subsequent formation of a 4-mercapto-N-phenylquinone monoimine (NPQM) layer. 2.7. Conjugation of Au-4ATP with Anti-LH-Ab (Au-4ATP-Antibody)

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To activate the free carbonyl groups formed on the gold surfaces, 0.2 M EDC and 0.05 M NHS solutions were prepared in MES buffer (pH 6.5). The AuNPs were mixed with these solutions for 1 h and covalent bonding with amine groups resulted in the formation of amine-reactive NHS esters on the surface of the NPs. Immobilization of antibodies occurred by submersing Au-4 ATP conjugate overnight in Tris-HCl buffer (pH 7.8), BSA and <0.1% sodium azide containing 25 μg mL−1 of antibody. The antibody-modified NPs were washed two times with MES buffer. Then, the nanoparticles were interacted with a mix of ethanolamine (10%, v/v) D-alanine (4 mgmL−1) solution for an hour to avoid non-specific interactions. The prepared Au-4ATP-Ab conjugate is performed microchip in order to achieve homogenous sandwich structure.

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2.8. Preparation of SERS-based sandwich immunoassay in the microchip

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Fe3O4@Au-Ab-LH conjugate was transferred to the first chamber of the four-chambered microchip and the modified AuNPs are transferred to the second chamber. Taking advantage of the magnetic properties of magnetite, the Fe3O4@Au-Ab-LH conjugate is collected with the aid of a magnet and dragged through the capillary valve of the microchip in the second chamber to interact with 4-ATP labeled AuNPs. This interaction resulted in the formation of a sandwich structure. Again, with the help of the magnet, the sandwich structure formed NPs were passed through the capillary valve and transferred to the third chamber where the washing buffer (0.05 M MES buffer) was present. In this case, the Fe3O4@Au-Ab-LH conjugation does not interfere with the dragging effect, eliminating the AuNPs carried in the third chamber, preventing access to the measuring chamber. At the end of the washing process, the particle was collected at a certain point by the magnet and transferred to the fourth chamber. The measurement was performed with SERS, and 4-ATP signals were recorded. The method was repeated using different LH concentrations and from these data, calibration graph was generated. The same method was performed with hCG, IgG and hGH to test the selectivity of the test.

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Here, functioning of the assay is mainly based on the performance of the capillary valves on the microfluidic chip. Once the solutions are transferred to the chambers through the access holes by pipetting, the liquid must not flow through the adjacent chamber, thus the next chamber could be filled with another liquid without mixing. This was achieved by designing the capillary valves to withstand the pressure generated during pipetting. It was calculated by using Surface Evolver [41] (an open-source software for analysis of interfaces under surface tension) that the valves could withstand pressure of 574 Pa. Since the chambers are mainly filled by capillary flow only, the pressure applied on the liquid was practically zero. Figure 1 presents the chip design, capillary valves, and operation of the chip.

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Figure 1. A. Exploded view of the microfluidic chip. B. As the first chamber is filled, the liquid gets pinned at the capillary valve indicated by the rectangle. C. Since the liquid in the first chamber was pinned at the capillary valve, second liquid can be dispensed in the second chamber without mixing with the first liquid. The 3rd and the 4th chambers are filled sequentially. D. Shape of the meniscus pinned at the capillary valve. Inlet pressure is 0 Pa. Meniscus shape was obtained by using Surface Evolver and overlaid with the capillary valve for illustration. E. Shape of the meniscus at the onset of breaching of the valve at 574 Pa inlet pressure. The pressure that the valve can withstand was computed by using Surface Evolver as described in [42]. (FI). Sequential filling of the chambers. It can be noted that mixing is only due to diffusion between the chambers. J. Magnetic nanoparticles moving between the chambers. Transfer of Fe3O4@Au NPs-Ab-LH conjugate to the first chamber and modified AuNPs with 4-ATP to the second chamber of the microchip. K. Derivation of the NPs in the second chamber and the formation of a homogeneous sandwich structure. L. Sandwich formation of NPs is dragged into the third chamber MES (pH 6.5) buffer. M. Transferring NPs to measurement chamber after washing step.

2.9. Validation of the developed method

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3. RESULTS AND DISCUSSION

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Within the scope of the validation study, selectivity, detection limit (LOD), limit of determination (LOQ), accuracy and recovery values were determined. To demonstrate the separation of endogenous and recombinant LH, control serum samples of BioRad origin containing LH at three different concentrations and LH concentration for Level 1, 2 and 3 were 1.5, 2.5, 5.0 IUL-1 respectively. The samples are divided into two parts; the first part was directly analyzed and the second part was analyzed and interacted with 2 IUL-1 LH. When the SERS spectra were examined, it was shown that the samples containing LH (2 IUL -1) had higher SERS intensities than the control spectra, and that the developed method was selective and sensitive in recognizing the target analyte. The regression equation of the calibration curve obtained from the control serum samples of BioRad origin containing LH between 0-250 IUL-1 was used to calculate the detection limit and detection limit values. LOD and LOQ values according to IUPAC were calculated from the following equation. LOD and LOQ were expressed as LOD = 3 × SD/b and LOQ = 10 × SD/b, where SD is the standard deviation of the response and b is the slope of the calibration curve. To calculate the accuracy, precision and recovery values, LH at concentrations of 0.4, 2.0 and 10.0 IUL-1 were added to control serum samples. The analysis was repeated under the same conditions for three days. Using the regression equation of the calibration curve obtained with original control serum samples, the results were evaluated.

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Rapid analysis and identification of biomolecules have recently been considered as an important research topic. For the detection of LH in the subclass of biomolecules, an alternative detection method with low detection limit, high sensitivity and high selectivity was developed. Here, we designed a rapid isolation and quantitative analysis method for human luteinizing hormone (LH) with labeled SERS probe. The system, immunomagnetic separation, and optical measurement technique were evaluated for the detection of LH from complex human serum matrix. Poly (methyl methacrylate) based microchip was used as a miniaturization device. After immunomagnetic separation, the homogeneous sandwich structure was formed in microchip and SERS measurements were recorded. Detailed characterization of NPs are given in our previous work [38].

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There are three main steps in the assay: (i) the immobilization of antibodies on Fe3O4@Au and Au NPs; (ii) isolation of LH from urine samples by Fe3O4@Au-MUA-Ab, and (iii) labeling the isolated biomolecules with AuNP-4ATP-Ab. We used 4-ATP as Raman label owing to its low toxicity and its ability to generate strong SERS signal [39,41]. It is a bifunctional molecule that can adsorb to the Au surface via the sulfur or nitrogen atom. The strongest band observed at 1086 cm−1 is assigned to C (benzene-ring)-S stretching vibration. The band at 1491 cm−1 is attributed to [ν(C–C) + δ(C–H)] mode. The SERS spectra show predominant enhancement of bands associated with C–S stretching and bending vibrations, indicating the binding through the S atom [43]. Surface roughness is essential for producing uniform and high-intensity spectra with the SERS method [44,45] and it was achieved by usage of NPs. In this study, we analyzed LH by recording 4-ATP intensity at 1086 cm-1 which was used as a marker in the detection of LH. Beside C-S stretching at 1086 cm-1, which is the most intense and characteristic band

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of 4-ATP, also peak at 1590 cm-1 is observed. The SERS spectra of LH samples spiked with different concentrations (0.4-250 IUL-1) are shown in Figure 2. As the concentration of LH increases, the intensity of the 4-ATP signal also increases. For each concentration, a sandwich structure was formed with the conjugates of Fe3O4@Au-LH-AuNP in microchip according to the steps of immunoassay method. There are two different linear calibration; which are obtained from LH spiked buffer and serum, respectively. In literature, calibration curves are generally generated for standard analytical specimens and calculations were made with these data [46,47]. A calibration graph of the band intensities of the C-S group at 1086 cm1 of the 4-ATP used as the Raman tag was established with values corresponding to the logarithms of LH concentrations. As a result, a linear calibration graph (R2 = 0.9923) was obtained from LH spiked buffer solution. On the basis of this linear calibration, the proposed assay enabled correlated SERS signal against increasing concentration of the analyte. Therefore, LH standards were spiked to control serum to demonstrate that the method is valid for real-sample experiments. The serum samples which are obtained from BioRad consist of 96 different analytes. As a result, a linear calibration graph (R2 = 0.9942) was obtained at these concentration range of 0.4 - 250 IU/L as shown in Figure 3. Based on the calibration curve obtained from serum sample, RSD values were also calculated. It showed that the sensitivity was not affected by the serum matrix and the proposed SERS assay was capable of analyzing LH in serum.

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Figure 2. SERS spectra with different concentrations of LH (0.4 -250 IUL-1) spiked in buffer (A) and serum samples (B) using sandwich based immunoassay method in the microchip.

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Figure 3. Calibration curves from SERS signals obtained from in MES buffer and in serum samples within the concentration of 0.4 – 250 IUL-1. The number of repetition of SERS measurement for each concentration is five (N=5).

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It is also possible that the protein binds directly to the surface of naked NPs in the suspension, however unspecific interactions and transport of these particles to the final chamber of the microchip will have an adverse effect on SERS signals. When the recorded SERS signals are examined; there was no significant difference between the observed signal and the blind signal indicating that it did not make a non-specific binding without LH antibody and did not show a homogenous sandwich immunoassay structure.

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LH has two polypeptide bonds; Α and β epitopes. The α-epitope of LH is similar to hCG, hFSH, hTSH. The β-epitope determines the biological and physiological properties of LH. In the designed immunoassay system, the LH-specific antibody should be used to capture the target protein from the environment. The antibody was tested with similar epitope proteins (hCG, IgG, hGH) found in human serum matrix to control LH selectivity. 0.5 mgmL-1 IgG and hGH in PBS buffer, this concentration being equivalent to 2 IUL-1 hCG and LH. The same procedure, designed for the LH assay, was also applied to existing proteins. At the end of the analysis, SERS signals for all proteins were recorded. According to the obtained results, it was determined that the proteins at the same concentration as LH were about values of SERS signal intensities with blind signal, LH antibody could recognize LH specifically, and other proteins did not interfere with LH assay shown in Figure 4. To calculate the accuracy and recovery values of the method, LH at concentrations of 0.4, 2.0 and 10.0 IUL-1 were added to control serum samples from BioRad. The analysis was repeated under the same conditions for three days. Using the regression equation of the calibration curve obtained with BioRad origin control serum samples, the recovery values were 90%, 93.5%, 96.3% and standard deviations were 3.8%, 1.1%, 3.9%, respectively (Table 1). These results in terms of accuracy and precision for biological sample analysis and these assays can be applied to serum samples for LH detection.

Table 1. Accuracy and precision of the assay (number of repetition, N=5) Homogenous Sandwich System Concentration (IU L-1)

Found (IU L-1)

Recovery (%)

RSD (%)

0.36

90.0

3.8073

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1.87

93.5

1.1243

10.0

9.63

96.3

3.9584

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RSD: Relative Standard Deviation

Figure 4. The SERS spectrum showing that the selectivity of the developed immunoassay.

To demonstrate the separation of endogenous and recombinant LH, control serum samples of BioRad origin containing LH at three different concentrations (LH concentration for Level 1, 2 and 3 respectively: 1.5, 2.5, 5 IUL-1) were used. The samples are divided into two parts; the first part was directly analyzed and the second part was analyzed and interacted with 2 IUL-1 LH. When the SERS spectra were examined, it

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was shown in Figure 5 that the samples containing LH (2 IUL-1) had higher SERS intensities than the blind used control sera, and that the developed method was selective and sensitive in recognizing the target analyte.

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Figure 5. SERS spectra obtained from the immunoassay using different urine samples before and after spiking with LH.

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During the formation of the sandwich structure, the aggregation of the particles is different from the batchtype systems due to the problem of clogging of the capillary connections of the resulting microchip [4850]. For this reason, a number of the nanoparticles to be placed in the chamber has been optimized. For this purpose, 5, 100, 150, 200 and 250 μL of Fe3O4@AuNPs was transferred to separate Eppendorf tubes, respectively, completed to final volume of 250 μL with deionized water. Washing steps and modifications was completed according to the procedure. The volumes were optimized correspond to 1.88 x 103, 2.82 x 103, 3.76 x 103 and 4.7 x 103 particle counts, respectively. As a result, the optimum amount of Fe3O4@AuNPs was determined to be 200 μL in a volume or 3.76 x 103 in particle amount. Since the loss in the washing step was neglected, the amount of Fe@AuNP injected into the microchip was approximately 150 particles. For the validation of the system, the regression equation obtained from BioRad origin control

serum samples were used. The LOD and LOQ values for the method were 0.0365 IUL-1and 0.1108 IU L-1, respectively. For LH determination, LODs were also compared to other methods (Table 2).

Table 2. Comparison of methods developed for LH determination compared to LOD values LOD

CL immunoassay

0.010 IU L-1 [51]

ECL immunoassay

0.100 IU L-1 [52]

NP labeled immunoassay

0.0012 IU L- [52]

Magnetic CL enzyme immunoassay

0.2 IU L-1 [16]

UPLC–MS

0.0001 IU L-1 [53]

Proposed method

0.036 IU L-1

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CL, chemiluminescence; ECL, electrochemiluminescence

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Analytical Method

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CONCLUSIONS

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Within the scope of this study, SERS-based sandwich immunoassay for fast and sensitive detection of LH by using antibody-functionalized magnetic nanoparticles and Raman dye-labeled nanorods is developed. If the modified nanoparticles are present in the modified form, in a single step (about 1 hour waiting time) by recognizing the target analyte due to the high selectivity of the nanoparticles modified with the antibody, the LH can be removed from the human liquid matrix. LC-MS, ELISA and gel electrophoresis (SDS-PAGE, IEF-PAGE, and SAR-PAGE) techniques used for protein analysis in the doping control centers require a lot of chemical and analytical processes. There is no need for pre-preparation, pre-enrichment, and immunopurification steps. The formation of the sandwich structure in the passive microchip and the recording of the measurements with the SERS technique requires about 20 minutes. The analysis is completed in less than two hours. The use of enzyme-labeled antibodies and secondary antibodies in the ELISA method and the use of fluorescence-labeled antibodies in fluorometric methods provides a great advantage as a cost compared with the pure monoclonal mouse antibodies used in this study. It has been shown that the developed method is more sensitive, selective, faster and less costly than the techniques used today in doping control analysis. Addressing the results obtained, we foresee that this work constitutes a platform for the application of passive microchips which can be produced commercially in the future and that it can be applied as an alternative method for LH determination in doping analyzes. Notes The authors declare no competing financial interest. Acknowledgments

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The authors would like to thank The Scientific and Technological Research Council of Turkey (TUBITAK) for their support on the project of COST CA15114-114Z783 as it has fundamentals of this study.

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BIOGRAPHIES Belma Gjergjizi received her Bachelor degree in 2015 in chemical engineering from Marmara University and her Master degree in 2017 under supervision of Prof. Dr. Necdet Saglam and Prof. Dr. Ugur Tamer in bioengineering from Hacettepe University. Her research interests focus on controlled drug delivery, bioanalysis, and biosensors.

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Ferah Çoğun received her Master degree in Mechanical Engineering in 2013 from Middle East Technical University. She has been conducting her PhD studies since 2014 under supervision of Prof. M. A. Sahir Arıkan at Department of Mechanical Engineering, Middle East Technical University. She is currently working as research assistant in Mechanical Engineering at Çankaya University. Her research interest focuses on the microchannel production methods.

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Ender Yıldırım received his BSc, MSc, and PhD degrees in mechanical engineering at Middle East Technical University, Ankara, Turkey in 2002, 2005, and 2011, respectively. From 2012 to 2013 he worked as postdoctoral researcher at Leiden Academic Center for Drug Research at Leiden University, the Netherlands. He is currently an assistant professor in Mechanical Engineering Department at Cankaya University, Ankara, Turkey, and the director of Microfluidics Design and Characterization Laboratory in the same department. His research interests include methods of platic microfluidic chip fabrication, capillary microfluidics, droplet-based microfluidics, Bio-MEMS, and biosensors.

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Merve Eryılmaz obtained her MSc degree in Analytical Chemistry at Faculty of Pharmacy, Gazi University in 2014. Her research was conducted on the development of new total protein assays via Raman spectroscopy. She is now pursuing her PhD degree under the supervision of Prof. Ugur Tamer at Analytical Chemistry Department at Gazi University. She is also working as a researcher in the same department and currently, she engages in surface-enhanced Raman scattering based analysis of biological specimens and related applications.

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Yeşim Selbes obtained her BSc degree in 2002 from Department of Biology, Osmangazi University, Turkey, and MSc degree in 2005 from Institute of Health Science, Osmangazi University, Turkey, and PhD degree in 2016 from Department of Nanotechnology and NanoMedicine, Hacettepe University, Turkey. She is a researcher in Turkish Doping Control Center, Hacettepe University, Turkey. Her research interests include bioanalysis, biosensor, nanomaterials, and nanotechnology, antidoping applications

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Necdet Saglam received his BSc degree in biology, MSc and PhD degree in biotechnology from Hacettepe University in 1979, 1982 and 1986, respectively. In 2000, he becomes a full professor at Hacettepe University. During 1997 - 2006 he was a vice-dean of the Faculty of Education at Hacettepe University. From 2007-2011, he worked as rector of Aksaray University. Now he is a full professor in the graduate school of science and engineering, department of nanotechnology and nanomedicine in Hacettepe University. Uğur Tamer received his PhD degree in 2003 from Hacettepe University Department of Analytical chemistry. Under the mentorship of Prof. Kadir Pekmez and Prof. Attila Yıldız, his research focused on modified electrodes and conducting polymers. He was involved in Prof. Harry Mark’s electroanalytical research group as a visiting scholar in the department of chemistry at Cincinnati University in 2002 and he had worked a post-doctoral researcher, working with Prof. Curtis Shannon, in the Department of Chemistry and Biochemistry at Auburn University in 2005. He was involved in Raman research group as a visiting

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professor, at the University of Maine, Lemans, France. His research focuses on modified electrodes, electrochemical controlled micro-extraction, enantiomer separations, magnetic and anisotropic nanoparticles, surface modification and surface-enhanced Raman scattering of the biological interface, bionanotechnology, and immunosensor based analytical devices. He is currently a professor of Pharmacy Faculty at Gazi University in Ankara, Turkey and he is a member of several nanotechnology-related COST actions.