Biothiol modulated growth and aggregation of gold nanoparticles and their determination in biological fluids using digital photometry

Journal Pre-proofs Biothiol modulated growth and aggregation of gold nanoparticles and their determination in biological fluids using digital photometry Elli Akrivi, Foteini Kappi, Vasiliki Gouma, Athanasios G. Vlessidis, Dimosthenis L. Giokas, Nikolaos Kourkoumelis PII: DOI: Reference:

S1386-1425(20)31316-0 https://doi.org/10.1016/j.saa.2020.119337 SAA 119337

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

8 November 2020 2 December 2020 8 December 2020

Please cite this article as: E. Akrivi, F. Kappi, V. Gouma, A.G. Vlessidis, D.L. Giokas, N. Kourkoumelis, Biothiol modulated growth and aggregation of gold nanoparticles and their determination in biological fluids using digital photometry, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020), doi: https://doi.org/10.1016/j.saa.2020.119337

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Biothiol modulated growth and aggregation of gold nanoparticles and their determination in biological fluids using digital photometry Elli Akrivi,1,2+ Foteini Kappi,3+ Vasiliki Gouma,3 Athanasios G. Vlessidis,3 Dimosthenis L. Giokas,3* Nikolaos Kourkoumelis1* 1

Department of Medical Physics, School of Health Sciences, University of Ioannina,

Greece 2 Neurology

Clinic, University Hospital of Ioannina, Greece

3

Department of Chemistry, School of Natural Sciences, University of Ioannina, Greece

+

These authors contributed equally.

*

Corresponding authors. Email: [email protected], [email protected]

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ABSTRACT This work describes a novel and easy to use method for the determination of biologically important thiols that relies on their ability to inhibit the catalytic enlargement of AuNP seeds in the presence of ACl4- ions and trigger their aggregation. UV-vis spectroscopic monitoring of the plasmon resonance bands of the formed AuNPs showed that the spectral and color transitions depend both on the concentration and the structure of biothiols. The colorimetric changes induced by biothiols were quantified in the concentration range from 5-300 μM in the RGB color system with digital photometry using a commercially available flatbed scanner as the detector. On the basis of these results, the applicability of the method was tested to the determination of glutathione in red blood cells and cysteine in blood plasma with satisfactory recoveries (88.7-96.5%), low detection limits (1.0 μM), good selectivity against major biomolecules under physiologically relevant conditions and satisfactory reproducibility (<8%). The method requires no technical expertise, is easy to apply and is performed without scientific equipment, holding promise as a simple test of biothiol testing even by non-experts.

Keywords: gold nanoparticles, biothiols, blood plasma, whole blood, instrumentationfree assay

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1. Introduction Biologically important thiols are ubiquitous components of biological fluids and are involved in a variety of biological and biochemical processes (e.g. regulation of cellular antioxidant defense system, protein biosynthesis, etc). The most abundant biothiol species are cysteine (Cys), homocysteine (HCy) and glutathione (GSH) and their concentration levels in biological fluids vary from a few μM to several mM. GSH is the most important biothiol in whole blood at concentration as high as 0.4-3.0 mM in red blood cells and at significantly lower concentrations in other biological fluids (<6 μM in human plasma and <1 μM in urine) [1,2]. On the other hand, the concentration levels of cysteine may vary from 135-300 μM in human plasma and 20-80 μM in urine while HCy levels exhibit a relatively uniform profile in biological fluids (<15 μM in either plasma or urine) [3-5]. Deviations from these “normal” levels has been identified in patients suffering from several diseases and clinical disorders which range in severity from hair loss and ageing to cardiovascular dysfunction, liver damage, osteoporosis and Alzheimer disease [5-8]. Due to the important biological implications of biothiols, there is a great interest in their determination in biological fluids. Instrumental techniques such as liquid chromatography, capillary electrophoresis and mass spectrometry provide accurate results and are well-suited for the simultaneous determination of individual biothiol species [6] and are widely applied in research laboratories. However, these techniques, although widely applied in research laboratories, have not be routinely used in ordinary biochemical or clinical laboratories in central or even more in decentralized healthcare units because they require specialized equipment and trained operators. Instead, thiolreactive probes and chemosensors based on colorimetric and fluorescence dyes have been widely used as an alternative to bulky instrumental techniques [7,8]. These probes

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are not selective to specific biothiol species but enable the fast determination of the total concentration of biothiols as an indicator of pathological conditions with high sensitivity, low analysis cost and ease of operation. Following the continued development in the field of nanomaterials science, a large amount of research has been devoted to the development of optical probes for the determination of biothiols using functionalized nanoparticle-based chemosensors and their features have been critically reviewed [9,10]. Noble metal nanoparticles are the most widely used scaffolds as they combine the strong affinity of the sulfhydryl group for noble metals with the strong (distance- and size-dependent) optical properties of metal nanoparticles. By virtue of these properties, many different sensing strategies have been developed that rely on the ability of biothiols to induce the aggregation or dispersion of noble metal nanoparticles through a variety of mechanisms that may involve the interaction of biothiols with receptor molecules on the nanoparticles surface, the replacement of stabilizing or functional molecules from the nanoparticles surface, or the disruption of interparticle bonds [9]. The apparent drawback of these methods is that nanomaterials must be either purchased (at a cost that cannot be disregarded) or synthesized and functionalized before use, a procedure that needs advanced nanotechnology skills as well as state-of-the-art equipment to characterize the nanomaterials. A simple way to overcome the use of pre-synthesized nanoparticles is to exploit the strong affinity of thiol-containing compounds for noble metals, in order to regulate the formation and growth of noble metal nanoparticles during their synthesis. Thiolcontaining molecules such as alkanethiols, mercaptans, thiolated polymers, and proteins have been used to regulate the growth and size of noble metal nanoparticles [11]. However, sensing strategies that use thiols both as target analytes and as regulators

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of nanoparticles formation and growth are still limited as compared to assays that use pre-formed nanoparticles. Willner and co-workers [12] and later Liao et al. [13] and Liz-Marzán and co-workers [14] employed this concept for the detection of organophosphate compounds by using thiocholine, produced from the enzymatic hydrolysis of acetylthiocholine by acetylcholine esterase (AChE), both as a reducing and a capping agent for AuCl4- and gold nanoparticles (AuNPs), respectively. A rather complex interplay between biothiols, silver ions and carbon dots were reported to promote the growth of silver nanoparticles on the carbon dots surface enabling the determination of biothiols in human plasma based on silver nanoparticle plasmon absorption [15]. We and others have showed that thiols-containing compounds can inhibit the photochemical reduction of AuCl4- to AuNPs thus enabling their determination either by observing the color transitions of the AuNPs suspension or by monitoring the kinetics of AuNP formation [16,17]. Those studies showed that the detection of thiols based on their ability to regulate the formation and growth of noble metal nanoparticles has several advantages over the use of pre-formed nanoparticles: a) the selectivity of the assay depends exclusively on the affinity of the metal ions with the sulfhydryl group. Hence, co-existing species may not interfere since they show little or no affinity for the metal ions. In contrast, pre-synthesized nanoparticles exhibit stronger non-specific interactions with non-target analytes due to the high surface area and chemical functionalization of nanoparticles that is necessary to ensure their stability, b) color transitions from the ionic to the elemental state are much more intense, as compared to the color transitions obtained when nanoparticles grow or are reduced in size, thus offering improved sensitivity and selectivity. Gold ions, for example, exhibit an absorption band at 323 nm while AuNPs typically exhibit their absorption

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maxima at λ>520 nm, c) there is no need to synthesize, characterize and stabilize the nanoparticles before use. In this work, we report a new two-step seeding growth method for the determination of biothiols that is based on their ability to act as growth-controlling agents of AuNPs . During the seed-mediated growth of AuNPs small metal particles (gold seeds) are used both as catalysts for the reduction of gold ions on their surface as well as nucleation points for the growth of particles. When biothiols are added into the gold seeds solution they bind on the AuNP seed surface affecting the growth and aggregation of AuNPs. In this manner, a concentration-depended colorimetric response and spectral transition is observed. Analysis is performed by recording the color transitions as light transmittance in a ubiquitous optical electronic device (i.e. a flatbed scanner) operating as a simple photometer. In this manner, the method combines a simple experimental procedure with consumer electronic devices as detectors thus enabling its application even by non-experts in resource-limited settings and noncentralized healthcare units. The method was tested for its applicability in the determination of major biothiol species in blood cells and plasma with satisfactory results.

2. Experimental 2.1. Chemicals Aspartic acid (Asp), L-cysteine (Cys), Glycine (Gly), Histidine (His), Lysine (Lys), Leucine (Leu), Alanine (Ala), Cystine (Cys-Cys), DL-homocysteine (HCy), glutamine (Glu), uric acid, magnesium chloride hexahydrate, sodium chloride, sodium sulfate, sodium hydrogen bicarbonate, sodium acetate, disodium hydrogen phosphate, potassium chloride, calcium chloride dehydrate, D(+)-glucose, and glacial acetic acid 6

were obtained from Sigma-Aldrich (Steinheim, Germany). L-Glutathione (reduced), Hydrogen tetrachloroaurate trihydrate (min. 99.9%), urea (>99%), bovine serum albumin (crystalline, 98%), and tris(2-carboxyethyl)phosphinehydrochloride (TCEP, 95%, 0.5 M) were obtained from Alfa Aesar (Karlsruhe, Germany). HPLC-grade acetonitrile was purchased from Fischer Scientific (Loughborough, UK). Nanosep® centrifugal vials with modified polyethersulfone membranes of 3 kDa molecular cut-off size were obtained from Pall Corp. (NY, USA). Nuclon (400 μL) 96-well microtiter plates with a clear flat surface that were purchased from Thermo Fischer Scientific (MA, USA).

2.2. Apparatus Absorbance measurements were performed in a Shimadzu UV-1800 dual beam spectrophotometer with matched quartz cells of 1 cm path length. IR spectra were recorded with a Perkin Elmer Spectrum Two attenuated total reflectance - infrared (ATR-IR) spectrometer. Transmittance (photometric) measurements were performed in a flatbed scanner (PerfectionV550 Photo, Epson) operating in transmittance mode by placing the microtiter plate between the imaging surface and the transparency unit of the scanner in order to align the white LED light source with the CCD strip detector establishing an optical path length [18,19]. During measurement (i.e. scanning in transmittance mode) the gamma function was set at 1.8 while all automatic correction functions embedded in the software (Easy Photo Scan, v.1.00.08, Epson) were disabled to ensure that the photometric data were not manipulated. Images of the microtiter plate were recorded as Joint Photographic Experts Group (JPEG) files at a resolution of 300 dpi. The color intensity was calculated as the mean gray intensity and the intensity of the color in the Red (R), Green (G) and Blue (B) channels, using the embedded

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functions of Image J software (US National Institutes of Health) in the original images without image processing. Dynamic light scattering (DLS) measurements were performed in a Malvern Zetasizer Nano ZS (Malvern Panalytical, Worcestershire, UK). 2.3. Preparation of the seed and growth solutions A 20 mL aqueous AuNP seed solution containing 0.25 mM HAuCl4 and 75 mM CTAB was prepared in a conical flask. Then, 0.6 mL of freshly prepared, ice-cold, NaBH4 solution (0.1 M) was added under stirring. The appearance of a pink coloration, upon addition of NaBH4 indicates the formation of AuNP seeds. The solution was kept in light-proof vials and used within 2-3 h after preparation. The preparation of the growth solution was performed by sequentially adding 1.6 mM CTAB and 12 mM ascorbic acid under continues stirring in 10 mL of aqueous HAuCl4 solution of 0.5 mM. The growth solution was prepared before use and stored for 30 min in a light-proof vial. 2.4. Experimental procedure For the determination of biothiols, 100 μL of seed solution was mixed with 250 μL of CH3COONa/CH3COOH buffer (pH=6). Then, 650 μL of sample and 1 mL of growth solution were added in tandem (with interim mixing). The solution was incubated for 10 min and an aliquot of 250 μL was placed in a 96-well microtiter plate. Analysis was performed by recording the color intensity of the light transmitted through the sample using a flatbed scanner operating in transmittance mode. The analytical signal was calculated as the difference in the color intensity of the blank and the samples in the blue channel of the RGB color system.

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2.5 ATR-IR characterization studies Aggregates of CTAB-AuNP-cyst assemblies were collected by the addition of 200 μM of cysteine to the seed solution and growth solutions (pH 6) and incubation for 1 hour at room temperature. The aggregates were separated from the supernatant by centrifugation and air-dried. The collected solid was milled and directly used for ATRIR analysis. The IR spectra of Cys and CTAB were also recorded as reference using the standard solids.

2.6. Analysis of real samples Artificial blood plasma (ABP) was prepared by mixing 137.5 mM sodium chloride, 4.2 mM sodium hydrogen carbonate, 3.0 mM potassium chloride, 0.5 mM disodium hydrogen phosphate, 0.5 mM magnesium chloride, 2.64 mM calcium chloride and 0.5 mM sodium sulfate in distilled water and adjusting the pH at the value of 7.4 [19]. Simulated blood plasma (SBP) was prepared as ABP but it also contained bovine serum albumin (35 g/L), glucose (5.0 mM), urea (3.0 mM), uric acid (220 μM) and a mixture of common amino acids found in blood plasma (0.5 mM of glutamine, glycine, valine, arginine, lysine and alanine; total concentration of 3.0 mM). Both ABP and SBP were spiked with 150-200.0 μM cysteine, which are within the representative cysteine levels in blood plasma [3-5]. A few ml of whole blood was collected from a group member voluntarily after informed consent was obtained. Since blood was not cultured or examined in any other way than the experimental protocol presented here, anonymity was ensured. Blood was taken in a designated room by a trained nurse of the university hospital. Red blood cells were separated from plasma after centrifugation at 800 g for 10 min. The collected red blood cells were lysed by 10-fold dilution with water, thorough mixing by vigorous 9

vortex-mixing and centrifugation at 10000g for 15 min [21]. The supernatant was collected and proteins were removed with centrifugal filters (MWCO = 3 kDa) at 12,000 g for 20 min at room temperature followed by precipitation with ACN (3:1 ACN: sample ratio) after centrifugation at 12,000 g for 10 min. The collected (supernatant) liquid was diluted with distilled water (total dilution approximately 200fold) and 650 μL were used for the determination of glutathione. The collected blood plasma (500 μL) was first treated with 1.0 mM TCEP for 30 min and interim vortex mixing to reduce free cystine to cysteine. Plasma proteins were removed as before, and the supernatant solution was collected and diluted 3-fold with distilled water (total dilution approximately 27-fold) and 650 μL were analyzed for its concentration in cysteine. For recovery studies, glutathione in red blood cell lysates and cysteine in blood plasma were added after protein precipitation in order to calculate the accuracy of the method avoiding potential losses during sample pretreatment (which was based on a standard procedure and was not investigated separately).

3. RESULTS AND DISCUSSION The sensing procedure we are reporting here stems from the earlier work on CTAB assisted seeded growth of AuNPs (i.e. gold nanorods) [22] but it exploits the strong affinity of thiols for gold in order to modulate the formation and growth of AuNPs. The principle of seed mediated synthesis of AuNPs relies on the ability of small AuNP seeds to serve as nucleation centers that catalyze the reduction of gold ions on their surface, leading to the controlled growth of gold nanorods [22]. In our approach, thiols bind on the surface of AuNP seeds, reducing the available catalytic surface, thus affecting the growth of AuNPs and inducing their aggregation. 10

To understand the sensing mechanism, we recorded the visible absorbance spectra of the solutions in the presence of increasing concentrations of thiols (Cys, HCy and GSH) after a fixed time interval of 10 min (Figure 1). Since the reaction time and the concentration of all reagents was maintained constant, the color transitions (red to blue) and the red shift in the absorbance spectra observed for all biothiols suggest that AuNPs aggregate with increasing thiol concentrations. Interestingly, the intensity of the colorimetric changes and spectral transitions are not the same for all biothiols but decrease according to the order: Cys > HCy > GSH. More specifically, the red shift in the visible absorbance spectra and the change in the color of the solutions becomes visible with the unaided eye at 3 μM for Cys, 10 μM for Hcy and 50 μM for GSH. The bathochromic shift of the absorption band and the appearance of a broad peak at longer wavelengths with increasing Cys concentrations is an indication that AuNPs form larger particle assemblies with anisotropic optical properties due to coupling of the surface plasmon resonance of adjacent AuNPs [23,24]. HCy, although structurally similar to Cys, induce less intense changes that involve blue shift of the absorbance spectra but without the appearance of broad peaks at higher wavelengths. Finally, the spectral changes induced by glutathione show a gradual bathochromic shift, but the changes were much less intense as compared to the other thiols at the same concentration levels. From the absorbance spectra we concluded that the aggregation of AuNPs is influenced both by the structure and the concentration of thiols. Firstly, thiols displace the protective coating from the AuNP surface (i.e. CTAB molecules here) and bind on the surface of AuNP seeds through strong covalent Au-S bonds. The complexation of thiols on AuNP seed surface reduce the available nucleation sites on the AuNP seed surface and consequently their ability to catalyze the reduction of gold ions that is responsible for AuNP growth. Since all experimental parameters and reagent

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concentrations were fixed, the red shift and color transitions observed at low thiol concentrations may be attributed both to AuNP growth. Conversely, at higher thiol concentrations, growth is inhibited and aggregation should be the main mechanism due the formation of zwitterionic networks through head-to-head interactions between the deprotonated carboxylate group of one thiol molecule and the protonated amine group of another thiol molecule [23-25]. The formation of thiol-mediated zwitterionic assemblies is verified by the IR spectra of CTAB-AuNP-Cys aggregates (Figure 2). Specifically, using 2nd derivative spectroscopy (Figure 2; inset), the weak stretching band S-H (2551 cm-1) of the free cysteine molecule was not observed in the CTABAuNP-Cys assembly suggesting that the SH group is a coordination site during the formation of the Au-cysteine complex, confirming the Au-S interaction. Furthermore, the symmetric stretching band of -COO- shifts from 1345 cm-1 in the cysteine molecule to 1400 cm-1 while the very weak bending bands of N-H at 1535 cm-1 shift to 1600 cm-1. Between 1000-1250 cm-1 the IR spectra of CTAB-AuNP-Cys shows some new bands probably corresponding to C-N stretching of the amine group. Another weak band at 3175 cm-1 corresponding to -NH3+ stretching shifts to 3370 cm-1 in CTAB-AuNP-Cys. These transitions are in good agreement with the vibrations of Cys and AuNP-Cys reported in previous studies [25]. The CTAB-AuNP-Cys assemblies also exhibit the characteristic vibrations of CTAB corresponding to C-N stretching at 720-730 cm-1, the stretching band of C-N+(CH3)3 at 910-930 cm-1, the stretching and bending bands of N+(CH3)3 at 3015 and 960 cm-1 respectively and the stretching bands of –(CH2)- at 2915 and 2850 cm-1 [26]. Interestingly, a new band appears in the CTAB-AuNP-Cys assembly at 775 cm-1 which is indicative of substituted C-H bending. Τhe thiol-mediated assembly of AuNPs through zwitterion-type interactions occurs for all aminothiols, yet, the colorimetric and the concurring absorbance spectra

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in our system are clearly differentiated for Cys, HCy and especially for GSH. The weaker effect of GSH on the aggregation of CTAB coated AuNPs, as compared to Cys and HCy, has been also reported in previous studies [25, 27] and can be attributed to the presence of a) the glutamate moiety, which exerts steric hindrance interactions [28], and b) the presence of an additional carboxylate group which is also deprotonated (pKa1=2.12 and pKa2=3.53) at the working pH value (pH 6); the carboxylate group may further inhibit the aggregation of glutathione-capped AuNPs by either interacting electrostatically with free CTAB molecules (i.e. thus increasing steric hindrance) [29] or by introducing a negative charge on the outer layer of AuNPs (i.e. electrostatic hindrance) [28]. These interactions, however, cannot explain why HCy behaves noticeably different than Cys, as both Cys and HCy are present as zwitterions in the working pH (5.02 and 5.62, respectively) and the only difference between them is the presence of an additional methylene group in HCy molecule. To shed more light on these interactions we performed DLS analysis of AuNPs in the presence of Cys, HCy and GSH. Although no conclusive evidence could be obtained because the reactions evolved over time and the average hydrodynamic diameter of the thiol-nanoparticle assemblies increased with reaction time, larger aggregates were clearly observed in the presence of Cys as compared to HCy and GSH. In the presence of Cys we also observed the presence of two different size distributions in the solution which in an indication of anisotropic growth of AuNPs [30]. We believe that the observed differences are related to the interaction of thiols with CTAB molecules based on the findings of earlier reports which have shown that cationic surfactant adsorption on thiol-modified gold surfaces increases as the fraction of methyl groups on the surfaces increase [31]. Therefore, as the packing density of surfactants (expressed as area per surfactant) at the surface of thiol-AuNPs increases

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with increasing thiol molecule size [31], the formation of large AuNP assemblies is impeded by steric hindrance interactions. Our notion is also supported by the facts that a) the aqueous solubility of the biothiols decreases according to the order: Cys (23.1 mg/mL) > HCy (14.8 mg/mL) > GSH (0.246 mg/mL) which agrees with the observed intensity of colorimetric and spectral changes in our system (i.e. less soluble compounds may be more effectively interact with the aliphatic chain of CTAB through hydrophobic interactions) [29,31], and b) the kinetics and intensity of aggregation decrease in the same order (Cys>HCy>GSH). Specifically, at concentrations higher than 50 μM of Cys, aggregates become visible with the unaided eye after 10-15 min. In contrast, no visible aggregates were observed for HCy and GSH under the same experimental conditions. In fact, the formation of aggregates in the presence of HCy was observed at longer incubation times while no aggregates were observed in the presence of GSH at all concentration levels examined (up to 200 μM) and irrespectively of incubation time. 3.1. Optimization of experimental conditions We optimized the experimental conditions of the assay by investigating the amount of growth and seed solution, which are necessary for the formation and growth of AuNPs, the kinetics of the assay by varying the reaction time and temperature and the pH of the solution which affects both the formation of AuNPs as well as the ionization state of cysteine. The seed solution is composed of small AuNPs prepared by mixing AuCl4- ions, CTAB as a stabilizer and NaHB4 as a reducing agent. The growth solution comprises of AuCl4ions and CTAB as stabilizer but uses ascorbic acid as a mild reducing agent which is too weak to reduce AuCl4- ions to AuNPs. The rate of AuCl4- reduction is enhanced only in the presence of gold seeds [22,32] therefore larger particles are formed by

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growth of the small particles (seeds). The optimum volume of seed and the growth solutions was investigated by varying the volume of the seed or growth solution univariately without changing the initial concentration of the individual components of each solution. In this manner, the concentration of each component changed simultaneously while maintaining the ratio between reagents concentrations constant in each solution. This approach was decided because nucleation and growth of AuNPs by the seeding growth method is affected not only by the concentration of the reagents but also by their relative abundance (i.e. ratio) in the solution. [22,24,32]. Hence, any differences were attributed to the properties of the seed or growth solution rather than the influence of each individual component. The optimum working conditions were selected based on the intensity of the net analytical signal that is calculated by subtracting the signal intensity obtained in the absence of biothiols (blank sample) to that obtained in the presence of biothiols. 3.1.1. Sequence of reactions Initially, we tested if the sequence of seeding growth reactions affect the analytical signal response by adding Cys, a) in the seed solution prior to mixing with the growth solution and b) in the growth solution prior to mixing with the seed solution. In the first occasion, Cys could complex with AuNP seeds and reduce their catalytic action in the growth of the AuNPs while in the second occasion cysteine could complex AuCl4- ions and inhibit their reduction on the AuNP seed surface. The most intense colorimetric changes were observed when Cys was mixed with the seed solution (prior to the initiation of the growth reactions) possibly because the complexation of thiols with AuNP seeds reduces the catalytic action of seeds. In contrast, the formation of Authiolate complexes could significantly suppress the reduction kinetics of Au ions [17]. Based on these observations, Cys was mixed with the buffered seed solution (to 15

maintain cysteine at a constant ionization state) prior to the addition of the growth solution. 3.1.2. Relative concentration of seed and growth solution The relative concentration of the seed and growth solutions were studied by varying their volumes (to the total volume of the solution) form 5%-50% (v/v) while maintaining all other parameters constant. Seed or growth solution volumes lower than 5 % (v/v) were not studied because the colors were too faint while volumes larger than 50% (v/v) (to the total sample volume of 2 mL) were considered impractical for the analysis of real samples as it would significantly decrease the sample volume that could be employed for the assay. Figure 3 shows the evolution of the net signal intensity in the mixture in the presence of variable concentrations of seed and growth solution. The results show that the signal evolves linearly with the volume of seed and growth solution and exhibits the maximum net signal response at the lower volume of seed solution (i.e. 5%, v/v) and the maximum volume of growth solution (i.e. 50% v/v). Considering that the net analytical signal is calculated as the difference in the color intensity in the presence and absence of Cys, these data can be explained by the fact that at low volumes of seed solution, Cys can interact with the surface of the seeds and inhibit their catalytic action in the growth of the AuNPs. At higher seed volumes an adequate amount of seed remains free in the solution and as a result their catalytic growth, and consequently the apparent colorimetric changes between the blank and the sample solutions, are less significant. Accordingly, low volumes of growth solution are not adequate to promote the growth of AuNPs and thus colorimetric changes are less intense. Based on these results the volumes of seed and growth solutions were fixed at 5% (v/v) and 50% (v/v), respectively.

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3.1.3. Effect of solution pH In the examined system, pH plays a diverse role in the generation of the analytical signal. In the first place, pH affects the interaction of CTAB with AuNPs and consequently the size and shape of the formed AuNPs [33,34]. In addition, pH regulates the interaction of cysteine with AuNPs [27,35,36]. The pKa values of cysteine are 1.96 and 8.18 for the carboxylic and amine groups respectively, suggesting that within this pH range cysteine will be present as zwitterion having both a negative and a positive charge. According to previous studies, cysteine molecules are attached on AuNPs surface through covalent Au-S bonds while the amino and carboxylate groups are involved in pH-depended electrostatic interactions [27,28, 30, 35,36]. In Figure 4, the maximum net analytical signal is obtained at pH=6. At this pH, Cys is present as zwitterion with equal positive and negative charge (pI=5.07) which favors the formation of inter-nanoparticle bridges between the -NH3+ and -COO- groups of Cys molecules

present on the surface of adjacent nanoparticles. As a result of this

interaction, AuNPs aggregate leading to red shift of the absorbance of the solution and to the appearance of a blue coloration. At lower pH values, –COO- is gradually protonated, thus electrostatic interactions are less significant, while CTAB molecules are densely packed on the seed surface inhibiting the deposition of Au ions and the growth of AuNPs [33]. At higher pH values, the CTAB layer becomes less dense (due to completion with OH- ions [37] and the collapse of micelle structure) [33], which leads to the formation of irregularly shaped AuNPs and a change in their optical properties (i.e. the solution turns from wine red to dark purple-red). Under these conditions the predominant Cys species is the mononegative anionic form (i.e. only the carboxylate moiety remains unprotonated); therefore, aggregation can only be induced by the formation of hydrogen bonds between the amine groups. The net difference in 17

color intensity in this occasion is reduced to almost 50% of that observed at pH 6. On the grounds of these observations, the reactions were performed at pH=6 using sodium acetate/acetic acid buffer. 3.1.4. Effect of time and temperature on the kinetics of the reaction The kinetics of the assay were evaluated by monitoring the evolution in the color of the solutions in the presence and absence of Cys by varying the reaction time (from 2 to 60 min) and temperature (from 20 to 50 oC). The results presented in Figure 5 show that the maximum net analytical signal is obtained after 30 min of reaction time at <30 oC. Therefore, analysis was performed after 30 min of incubation at 20 oC. 3.2. Investigation of selectivity The selectivity of the method against other species was evaluated by comparing the net signal intensity of Cys in the presence and absence of major biomolecules typically found in biological fluids (such as amino acids, sugar, antioxidants, inorganic electrolytes etc) at concentrations equal or higher than the physiologically relevant concentration levels in human serum [38]. Based on the results presented in the bar plots of Figure 6a. we concluded that the presence of common biomolecules (such as amino acids, glucose, ascorbic acid, uric acid, urea, and creatinine) do not interfere with the determination of cysteine. From the same results it can also be inferred that oxidized biothiols (such as cystine) do not affect the formation and growth of AuNPs therefore the method could conceivably be used to discriminate between reduced and oxidized biothiol species. We attributed the lack of interference to the protective CTAB layer of AuNPs which protects the AuNPs from interacting with the other biomolecules while thiols, due to their high affinity for Au, can displace CTAB from the Au seed surface and complex through covalent Au-S bonds. 18

To study the effect of inorganic electrolytes, we prepared a series of calibration curves in ABP and distilled water containing 10-100 μM Cys and compared the slopes of the curves. We observed that the net analytical signal and the slope of the calibration curve at physiologically relevant NaCl concentrations (i.e. 150 mM NaCl) was higher than that obtained in distilled water (0.29 in ABP and 0.23 in distilled water, respectively). This was attributed to the aggregation of AuNPs which leads to more intense red shift of the absorbance spectra. The enhancing effect of NaCl on the aggregation of CTABcoated AuNPs has been also reported before [26]. This positive interference could be mitigated by reducing the concentration of NaCl to 15 mM, corresponding to a minimum of 10-fold dilution of the initial sample (considering that the physiologically relevant NaCl concentrations is close to 150 mM). As show in Figure 6b, the calibration curve obtained in 10-fold diluted ABP produced similar analytical signals and slope (i.e. 0.22) to those obtained in distilled water. 3.3. Analytical figures of merit The performance of the assay to the determination of different biothiols was evaluated using standard solutions of Cys, HCy, and GSH which are the most abundant biothiols species in biological fluids (glutathione in whole blood, cysteine and homocysteine in blood serum and urine). For Cys an extended dynamic range could be achieved from 3300 μM using a logarithmic function while for HCy and especially GSH, which produced less intense color transitions, linear calibration functions could be used to correlate the analytical signal to the concentration of each thiol species (Table 1). The repeatability (calculated as the relative standard deviation of five replicate measurements) ranged from 1.8% to 7.7% at 10 μM and 3.6% to 8.3% at 100 μM, which was satisfactory. The detection limits (calculated as the signal of the blank plus three times the standard deviation of the blank) were 1 μM for Cys and 3 μM for HCy, and 19

GSH. Although other assays that employ more sophisticated methods (e.g., colorimetric or fluorescence methods based on nanoparticle sensors) have been reported to accomplish the determination of biothiols at lower concentrations (Table 2), the detection limits and the linear range of the developed method are well below the concentration of total biothiols in biological fluids. 3.4. Analysis of real samples Before the analysis of real samples, we evaluated the applicability of the method in artificial body fluids in order to assess potential matrix effects under biologically relevant conditions and identify the appropriate sample pre-treatment conditions for analysis. ABP was first used as model matrix which is a mixture of inorganic salts without other biomolecules. We observed that the best results were obtained when the samples were diluted by 20-fold or more so that a) the ionic strength of the solution is lower than 8 mM and b) the concentration of cysteine falls within the first part of the logarithmic dose-response curve where the analytical signal increases linearly with cysteine concentration (3-15 μM). Therefore, by diluting the samples (by at least 20fold) good recoveries could be obtained (Table1). The observed dilution is higher than that observed previously in the presence of NaCl only, suggesting that other inorganic electrolytes may also affect the aggregation of AuNPs. Basing on the results obtained with ABP, we investigated the optimum procedure for treating simulated blood plasma, which is composed of ABP enriched with common blood components at physiologically relevant concentration levels (see the experimental part for details). We investigated the optimum conditions for a) reducing oxidized thiols (i.e. cystine), which constitute the major biothiol species in human plasma, and b) precipitating proteins, which carry a significant amount of 20

biothiols (e.g. albumin contains 16 cysteine units per molecule) and may interfere with the analysis producing false-positive results. Through trial-and-error tests with SBP, we found that the maximum TCEP concentration in the final solution, that could be tolerated without affecting the growth and formation of AuNPs is <0.05 mM. At higher concentrations, a positive interference was gradually observed because an excess of residual (unreacted) TCEP could reduce the gold ions in the growth solution. Therefore, the reduction of cystine to cysteine was performed before protein precipitation with 1.0 mM of TCEP as reducing agent for 30 min followed by dilution to regulate the concentration of TCEP in the final extract. Protein removal before analysis was achieved with modified polyethersulfone membranes (3 kDa Nanosep, Pall Co.,) at 12,000 g with 20 min followed by precipitation with ACN at a 3:1 ratio after centrifugation at 12,000 g for 10 min. On the grounds of the observations made above, the collected (protein-free) extract was further diluted by 3-fold with distilled water so that a final dilution of at least 25-fold of the raw sample was achieved. The recoveries obtained by this procedure were 94-111% which were deemed as satisfactory (Table 3). Therefore, the method was further tested to the analysis of real blood samples. The determination of biothiols in biological fluids was performed by determining the concentration of GSH in red blood cells and Cys in blood serum. For further validating the method, we also spiked the samples with known amounts of GSH and Cys and analyzed them again to calculate the recoveries of each biothiol species (Table 3). These experiments show that the measured recoveries lie between 88.7% and 96.5%, indicating the trueness of the assay and its suitability for routine biochemical analysis of biothiols in biological fluids.

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4. CONCLUSIONS In this work we demonstrated that low molecular weight thiols can inhibit the seed mediated growth of AuNPs and induce their aggregation in a concentration-depended manner. Furthermore, the intensity of colorimetric transitions and spectral changes were found to depend on the structure of thiols and their interaction with CTAB molecules that were used as a protective layer of AuNPs. The color transitions were recorded in a flatbed scanner in transmittance mode enabling high quality photometric measurements to be performed using ubiquitous electronic devices without instrumental detectors. Based on these findings we developed a simple colorimetric assay for the sensitive determination of biothiols at the low μM levels and demonstrated its utility to the analysis of biothiols in biological fluids.

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nitrobenzoxadiazole

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fused sulfide,

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Figure 1. Visible spectra and the respective colorimetric changes (inset images) observed with increasing concentrations of Cys, HCy and GSH (0-200 μM). Other experimental conditions: 100 μL seed solution, pH=6, 1 mL growth solution, reaction time: 10 min. To facilitate color perception, a magnified view in batlow color map of Figure 1 is given in the Supporting Information.

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Figure 2. ATR-IR spectra (average data of 5 replicates) of cysteine (blue line), CTAB (red line) and CTAB-AuNP-Cys assemblies (green line). The inset depicts the 2nd derivative spectra within the range of 2480-2600 cm-1.

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Figure 3. Effect of the relative concentration of the seed and growth solutions in the analytical signal response of Cys. Experimental conditions: pH=6, reaction time: 5 min, Cys=100 μM. Error bars represent standard error calculated for triplicate samples.

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Figure 4. Effect of solution pH on the color signal intensity of Cys. Experimental conditions: 100 μL seed solution, 1 mL growth solution, reaction time: 5 min, Cys=100 μM. Error bars represent standard error calculated for triplicate samples.

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Figure 5. Influence of reaction time and temperature on the colorimetric signal response of Cys. Experimental conditions: 100 μL seed solution, 1 mL growth solution, pH 6, Cys=100 μM. Error bars represent standard error calculated for triplicate samples.

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Figure 6. Selectivity of the assay against (a) common biomolecules and (b) inorganic electrolytes at concentrations equal or higher than the physiological levels (i.e. amino acids: 1.0 mM, glucose: 5.0 mM, urea: 3.0 mM, NaCl: 150.0 mM). The control sample in Fig. 6a contains only Cys while all other samples contain Cys and the potentially interfering biomolecule. Error bars represent standard error calculated for triplicate samples. The linear curves in Figure 6b are the result of linear regression.

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Table 1. Analytical figures of merit of the assaya. Cysteine (Cys) Dynamic range: 3-300 μM (y=13197 lnx - 8500, R2=0.998) LOD=1.0 μM RSD=7.7% (10 μΜ), 3.8% (100 μM), n=5

a

Homocysteine (HCy) Linear range: 10-300 μM (y=186.7x + 20830, R2=0.994) LOD=3.0 μM, RSD=5.7% (10 μΜ), 8.3% (100 μM), n=5

Glutathione (GSH) Linear range: 5-300 μM (y=405x + 1465, R2=0.99) LOD=3.0 μM, RSD=1.8% (10 μΜ), 3.6% (100 μM), n=5

The concentrations that were used to calculate the calibration curves were 0, 1, 3, 5, 10, 30, 50, 100, 200, 300 μM for each of the biothiols species. The inset

graphs show a digitally cropped image (to facilitate the aspect view) of the sensing zones (micro titter plates). The first plate is the control (blank) sample while the marked area (dot lines) indicates the working range of the method for each biothiols species. A circular area occupying 80% of the total sensing area was used to measure the analytical signal which is recorded in the blue color region of the RGB color system.

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Table 2. Comparison of the method with recent methods for the determination of biothiols. Detection technique

Material or

Real samples

Linear range

LOD

Recoveries (%)

Reference

(μM) a

(μΜ)a

Spiked artificial body fluids

15-200

15

95,6-97,3

16

Turbidimetry / Colorimetry AgCl / AgNPs

Blood plasma

10-100

8.1

92-97

18

Fluorescence

Spiked blood plasmab

5-200

0.3

98.6-111.5

39

Colorimetry / Fluorescence RhB-MnO2 NFs

Spiked blood plasmab

0–15

0.14

89.3-116.3%

40

Fluorescence

HD-NBD

Imaging of MCF-7 cells

1-45

0.36

-

41

Fluorescence

Fluorosceine-AuNPs

Spiked blood plasmab

0.04-1.0

0.027

95.6-103.9

42

Visual / Colorimetric

AuCl4- / AuNP seeds / AuNPs

Whole blood, blood plasma

3-300

1.0

88.7-114

This method

sensing element Colorimetric / Visual

AuCl4- / AuNPs

VS-CDs

PDA-AuNPs: polydopamine (PDA)-functionalized gold nanoparticles (AuNPs), HD-NBD: etrahydro[5]helicene fused nitrobenzoxadiazole, VSCDs: Vinyl sulfone - carbon dots, RhB-MnO2 NFs: Rhodamine B-manganese dioxide nanoflakes. a Linear ranges and LODs may vary for individual biothiols. b Biothiols were determined after spiking of known concentrations in pretreated (e.g. diluted, etc) serum.

36

Table 3. Application of the method to the analysis of biothiols in artificial and real body fluids and recoveries from spiked samples. Sample

Measured (μΜ) a

Spiked (μΜ) b

Found (μΜ)

Recovery (%)

Artificial blood plasma

0

150.0

172±8.0

114±4.6

0

200.0

198,0±5.1

99±2.5

0

150.0

141±9.3

94±6.6

0

200.0

220±12.1

111±5.5

1440±120

250.0

1640±140

96.5±8.5

500.0

1825±190

92.0±10.4

50.0

215±19

88.7±8.8

100.0

268±18

90.3±6.7

Simulated blood plasma

Red blood cells

Blood plasma

186±14

a

Values refer to undiluted samples. b Artificial, simulated, and real blood plasma were fortified with cysteine while red blood cell extracts were fortified with glutathione.

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Graphical abstract

38

HIGHLIGHTS 

Biothiols selectively affect the growth and aggregation of gold nanoparticles



Color and spectral changes depend both on structure and concentration of biothiols



Biothiol-induced color changes are monitored with a scanner as photometric detector



Easy-to-use method with minimum technical expertise and no scientific equipment

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CRediT author statement E. Akrivi: Investigation, Validation, F. Kappi: Methodology, Investigation, V. Gouma: Methodology, Investigation, A. Vlessidis Data Curation, Visualization, D.L. Giokas: Conceptualization, Investigation, Formal analysis, Writing - Original Draft, N. Kourkoumelis Conceptualization, Formal analysis, Writing - Review & Editing.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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