In situ hydrazine reduced silver colloid synthesis – Enhancing SERS reproducibility

Accepted Manuscript In situ hydrazine reduced silver colloid synthesis – enhancing SERS reproducibility Vera Dugandžić, Izabella Jolan Hidi, Karina Weber, Dana Cialla-May, Jürgen Popp PII:

S0003-2670(16)31201-6

DOI:

10.1016/j.aca.2016.10.018

Reference:

ACA 234841

To appear in:

Analytica Chimica Acta

Received Date: 19 June 2016 Revised Date:

13 September 2016

Accepted Date: 11 October 2016

Please cite this article as: V. Dugandžić, I.J. Hidi, K. Weber, D. Cialla-May, J. Popp, In situ hydrazine reduced silver colloid synthesis – enhancing SERS reproducibility, Analytica Chimica Acta (2016), doi: 10.1016/j.aca.2016.10.018. 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.

ACCEPTED MANUSCRIPT In situ hydrazine reduced silver colloid synthesis – enhancing SERS reproducibility

Vera Dugandžića,b, Izabella Jolan Hidia,b, Karina Webera,b, Dana Cialla-Maya,b* and Jürgen

a

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Poppa,b

Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University

Jena, Helmholtzweg 4, 07743 Jena, Germany

Leibniz Institute of Photonic Technology, Albert Einstein Straße 9, 07745 Jena, Germany

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b

[email protected] +49 (0)3641-206309

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*Corresponding author:

Herein we report a novel strategy for the in situ synthesis of the silver colloids for LoC-SERS

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applications. Silver nanoparticles are obtained in a segmented flow based glass microfluidic chip by the reduction of silver ions with hydrazine in ammonium hydroxide solution. Citrate ions are used as protecting agents. The synthesized nanoparticles are characterized by UV-

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VIS spectroscopy, SEM and TEM imaging. The SERS performance of the in situ synthesized nanoparticles is tested by using adenine as a test analyte right after the colloid synthesis.

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Reproducibility is tested by repeating the measurements three times at independent days applying the same measurement conditions. In comparison with nanoparticles synthesized in a conventional strategy i.e. in a large batch, chip synthesized nanoparticles show a better dayto-day and long-term reproducibility, lower detection limits and broader working ranges. The great advantage offered by the in situ synthesized colloids combined with the already proven potential of LoC-SERS for bioanalytics, raises the possibility of the employment of LoCSERS as a fast and sensitive analytic tool in a plethora of applications.

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ACCEPTED MANUSCRIPT Keywords: LoC-SERS, microfluidic, in situ, silver colloid, adenine

1. Introduction

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Modern analytical chemistry offers highly reliable and accurate methods for analysis of basically any analyte, whereby chromatographic methods are the most widely spread

techniques.[1, 2] Even though these analytical methods have high precision and low detection

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limits, they are often very expensive, time consuming, require complex sample preparation and trained personnel. Therefore, there is an excessive need for new, fast, simple and cost

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effective analytical procedures which will allow fast and reliable routine analysis for a vast variety of applications, covering environmental protection, analytical quality control, food safety, medical application and many others.

Surface Enhanced Raman Spectroscopy (SERS) was demonstrated to be a promising tool

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which can fulfill all the criteria named above. By combining the high specificity of Raman spectroscopy with the high sensitivity provided by the enhancing effect of plasmonic materials, SERS allows the detection of trace amounts of analytes with molecular

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specificity.[3] Furthermore, SERS gives the possibility for analyses in aqueous solutions[4], which makes it a perfect method for direct detection of medically relevant analytes from the

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physiological fluids without excessive sample preparation. Within the last years, medical applications of SERS gained much attention in the scientific community[5-7] e.g. drug monitoring[8, 9], SERS based enzyme assays[10-12] and SERS based detection of pathogens.[13] The SERS effect is observed when the analyte is bound to a suitable plasmonic material or in its close proximity.[14] Thus, plasmonic materials have crucial importance for any SERS based analytical procedure. Silver and gold nanoparticles are the most commonly used plasmonic materials, since their plasmon frequencies are in the visible region of the electromagnetic spectrum.[15] Colloidal gold and silver nanoparticles are in general easy to prepare by a simple 2

ACCEPTED MANUSCRIPT chemical reduction of a metal precursor in the presence of a protective agent which is used to stabilize the suspension. Nanoparticles of different size and shape and with different capping agents were synthesized and were tested for their SERS activity.[16-18] It was recently shown that no SERS enhancement is observed from non-aggregated spherical nanoparticles.[19]

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Therefore, to obtain SERS spectra with high signal-to-noise ratio, it is necessary to aggregate nanoparticles to create hotspots. Colloid based SERS measurements are typically performed in cuvettes. However, it is shown that the signal intensity decreases over time. This is the

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consequence of the aggregation and deposition of the particles on the bottom of the cuvette. These two contributions make cuvette based SERS measurements highly unreproducible. For

avoid precipitation of the colloids.

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consistent measurement conditions it is necessary to provide constant mixing conditions to

Additional issues related to colloid based SERS are associated with nanoparticle synthesis showing poor reproducibility, since even a slight change of the reaction parameters can affect

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the size and shape of metallic nanoparticles. An additional shortcoming is an observation that colloids are changing over time e.g. by aggregation or precipitation, thus giving different SERS activities. Therefore, a comparison between the SERS data obtained in different

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measurements employing colloids is challenging. Various strategies are applied to overcome this difficulty, whereas the incorporation of an internal standard in the nanoparticles seems to

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be the most promising approach.[20]

Employment of microfluidic platforms in combination with SERS opened the door for Labon-a-chip SERS (LoC-SERS).[21, 22] Following LoC-SERS measurement procedures, analyte, colloid solution and the aggregation agent are mixed directly in the microfluidic chip, using high precision syringe systems, while the SERS spectra are collected upon mixing in one of the channels. Microfluidic platforms assure constant conditions of the colloid and analyte dosing. The measurement can be performed using minimal amounts of the sample. High throughput of the measurements in a microfluidic setup provides a possibility for statistical 3

ACCEPTED MANUSCRIPT evaluation of the data[23], thus making the results more reliable and more comparable. Segmented flow microfluidic devices showed many advantages for the SERS measurement compared to continuous flow microfluidic devices.[24] The biggest advance is the absence of the memory effect as well as constant mixing conditions for the colloid and analyte mixtures.

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Moreover, the possibility of automatization improves the potential toward an online monitoring application of such a setup.

High precision of dosing of the solutions together with uniform and constant mixing offered

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by the segmented flow microfluidic setups will allow for the in situ synthesis of colloids. As a consequence, each droplet contains a new batch of freshly prepared colloid. This strategy

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should help to enhance the reproducibility of the colloid synthesis since it provides constant conditions. Furthermore, since each SERS measurement is performed with a freshly prepared batch of colloids, aging of the colloids becomes irrelevant. This strategy was successfully applied by Gao et al. in 2014 for the real-time SERS analysis of diquat dibromide in water [25],

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using the Leopold-Lendl protocol for the nanoparticle synthesis.[26] Furthermore, optical fibers can be coupled with microfluidic devices, raising the possibility for the in situ characterization of the particles inside the microfluidic channel[27], which is

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very interesting feature for the nanoparticle synthesis in microfluidic platforms. Herein, we present a novel method based on the hydrazine reduction of silver in ammonium

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hydroxide solution for the in situ preparation of the silver colloid in a segmented flow directly before the SERS measurement. Adenine was used as a test analyte since this molecule is known as a good Raman reporter. As compared to the approach of Gao et al. our approach was designed to be applicable for in situ nanoparticle synthesis in already well established glass chip platform[28], where Leopold-Lendl protocol cannot be used due to the damage to the inner walls of the glass chip caused by the sodium hydroxide solution. Data collection applied allowed more advanced statistical treatment of the data, making final output and comparison of the data acquired in separate measurements more reliable. Furthermore, short term and long 4

ACCEPTED MANUSCRIPT term reproducibility was tested. This approach results in the improved reproducibility of the measurements compared to the batch prepared nanoparticles. Furthermore, lower detection limits as well as broader working range is observed. Together with the possibility of widening the working range simply by increasing the amount of the precursors, which results in the

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formation of larger number of the nanoparticles, and consequently a larger surface available for analyte adsorption, this approach shows great advantage over nanoparticle synthesis for

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SERS in large batches.

2.1. Materials and instruments

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2. Experimental

Silver nitrate (99,9999%), hydrazine hydrate (98%), ammonium hydroxide solution (5.0 M)

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and mineral oil were purchased from Sigma-Aldrich. Trisodium citrate dihydrate, ammonium hydroxide solution (32 %) and potassium chloride (≥99 %) were purchased from Carl Roth.

water.

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All chemicals were used without any purification. All solutions were prepared with Milli-Q

UV-Vis spectra were recorded applying Jasco V-670 spectrophotometer and Analytic Jena

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Specord 250 spectrophotometer. SERS spectra were collected with a WITec Raman microscope (WITec GmbH, Ulm, Germany) equipped with a continuous wave diode-pumped solid-state laser with an excitation wavelength of 514 nm, using Zeiss EC “Epiplan” DIC, 20x, N.A. 0.4 objective in backscattering configuration. A grating of 600 lines per mm was employed with a spectral resolution of ~5 cm-1. A thermo-electric cooled CCD camera with 1024x127 active pixels and pixel size of 26x26 μm was used for the detection. TEM images were performed on a FEI Technai G² 20 system by blotting 15 µL of the nanoparticle solutions onto carbon coated TEM grids (Quantifoil, Germany) at an acceleration 5

ACCEPTED MANUSCRIPT voltage of 200 kV. SEM images were performed on a Zeiss (LEO) 1530 Gemini fieldemission scanning electron microscope. The solutions were deposited onto a conductive substrate and coated with a thin layer of gold by sputter evaporation.

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2.2. Silver nanoparticle synthesis

Silver nanoparticles were prepared in a reaction between silver nitrate and hydrazine hydrate

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in ammonium hydroxide solution, employing trisodium citrate as a protective agent. In detail, two solutions were prepared. Solution A contained 2 mmol L-1 silver nitrate, 0.8 mmol L-1

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sodium citrate and 18 mmol L-1 ammonium hydroxide. Solution B contained 1 mmol L-1 hydrazine hydrate. The solutions were stored in the fridge at 5 °C after preparation and were tempered to room temperature before the nanoparticle synthesis.

For the synthesis in a large batch, 5 mL of solution B was added to 5 mL of solution A and

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mixed. Colloid formation occurred in around 10 seconds after mixing. The in situ synthesis of the silver nanoparticles was pursued in a microfluidic platform which has been previously described (Figure 1).[28] Solutions A and B and the separation medium

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(mineral oil) were filled in 60 mm glass syringes (ILS GmbH) and connected to the microfluidic chip using teflon capillary tubes with inner diameter of 0.5 mm. All chip ports

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were first filled with carrier oil which was pumped with a flowrate of 10 nL s-1. Subsequently, solutions A and B were pumped into the stream of carrier oil through the ports 1 and 2, respectively (Figure 1), both with the flowrate of 7 nL s-1, forming droplets. The flowrates of the carrier oil and of the solutions A and B were controlled by a high precision neMESYS syringe pump system (Cetoni GmbH) and were kept constant during the entire procedure. The nanoparticle solution was collected at the waist outlet of the chip and used for the characterization of the nanoparticles, while all unused ports of the chip were blocked. To investigate the actual shape of the particles in the measurement site, an additional experiment 6

ACCEPTED MANUSCRIPT was performed, in which a 5 g L-1 solution of polyvinylpyrrolidon (PVP) was pumped through port 4 during the synthesis procedure, thus, wrapping particles with a polymer layer preventing them from further change in shape. Synthesized nanoparticles were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM) and transmission electron

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microscopy (TEM) (Figure 2).

Figure 1. A) Scheme of the microfluidic chip used for the colloid synthesis and LoC-SERS measurements. B)

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Block scheme of the in situ silver colloid synthesis for the LoC-SERS measurements. Labels on the block scheme coincide to those on the microfluidic chip scheme. For the nanoparticle characterization, nanoparticles were synthesized in a microfluidic chip and collected at the waste outlet avoiding steps of analyte addition and

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SERS measurement. Unused ports were blocked.

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2.3. LoC-SERS measurements

To test the SERS performance of the synthesized colloids, adenine was used as a model analyte. For the LoC-SERS measurements of adenine with in situ prepared nanoparticles, solutions A and B were pumped into carrier oil stream (10 nL s-1) through the ports 1 and 2 (Figure 1) of the microfluidic chip with flowrates of 7 nL s-1. Adenine solutions of various concentrations and 0.2 mol L-1 KCl were pumped through ports 4 and 5 with a flowrate of 6 nL s-1 and 5 nL s-1, respectively. The unused port was blocked. 7

ACCEPTED MANUSCRIPT For the LoC-SERS measurements of adenine with batch prepared nanoparticles, the colloid solution was pumped through port 1 (Figure 1) with a flowrate of 14 nL s-1 while the adenine solutions and 0.2 mol L-1 KCl were pumped through ports 4 and 5 with the same flowrates as in the previous case. Unused ports were blocked.

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LoC-SERS spectra were collected continuously with an integration time of 1 s with a focus fixed in the middle of the third channel of the microfluidic chip. The laser power used was 35

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mW. For each concentration of adenine 600 spectra were recorded and statistically processed.

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2.4. Data processing

Data processing was conducted using an in house developed algorithm in the programming language R.[29] Since the spectra were recorded each second at a fixed point in the third channel of the microfluidic chip, the large data set contained Raman spectra of the carrier

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medium oil and SERS spectra of adenine collected in droplets. Therefore, the first step in the data processing was the removal of the oil spectra followed by the removal of the spectra containing cosmic spikes. Adenine spectra were further grouped in stacks of three (which

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represents the spectra obtained in one droplet), averaged and cut to the desired spectral region. Background correction was performed by the selective nonlinear iterative clipping (SNIP)

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algorithm.[30] Univariate statistical analysis was performed, calculating the integrated peak area of the band around 730 cm-1, which corresponds to the ring breathing mode of adenine. For the peak integration, Simpson’s rule was applied. The integrated peak area is plotted as function of the concentration.

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ACCEPTED MANUSCRIPT 2.5. Reproducibility

Three independent measurements are applied to show the reproducibility of the LoC-SERS measurement of adenine concentration with in situ synthesized nanoparticles. Two

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measurements were done in two consecutive days using the same A and B solutions for the nanoparticle synthesis. Third measurement was done after two months with newly prepared solutions A and B. The equivalent reproducibility test in two consecutive days was done using

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the batch prepared nanoparticles. The measurement after two months was not performed due to the visible aggregation of the batch prepared colloids. Data obtained during two

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consecutive days were compared and the limit of detection (LOD) and the limit of quantification (LOQ) values were calculated using Equation 1 and 2 for the obtained data sets: LOD = PAblank + 3 ⋅ σ blank

(2)

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LOQ = PAblank + 10 ⋅ σ blank

(1)

with PA being the peak area of the blank and σ representing the standard deviation of the

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blank.

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3. Results and discussion

The reaction applied for the silver nanoparticle synthesis in this study can be described by the chemical equation

4[Ag ( NH 3 ) 2 ]NO3 + N 2 H 4 + NH 4OH → 4 Ag + N 2 + 4 NH 4 NO3 + 4 H 2O

(1)

Diamminesilver(I) complex ions were used as a silver source. The formation of this stable complex ion, due to addition of ammonium hydroxide to the silver nitrate solution, seems to be of high importance for the reaction, since it allows a kinetic control over the reaction. An 9

ACCEPTED MANUSCRIPT increase of the ammonium hydroxide concentration results in a decrease of the reduction potential of silver, from 0.7993 V for the simple Ag+ ion in the water solution[31] to the 0.373 V for the [Ag(NH3)2]+ ion[32], leading to the decrease of the reaction rate. This fact is of high importance for the in situ nanoparticle synthesis, since it prevented clogging of the chip

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channels by silver due to the delay in the reaction occurrence. Furthermore, ammonium

hydroxide stabilizes the silver(I) solution; thus, giving the opportunity to store the solution for a longer time as compared to a silver(I) solution that does not contain ammonium hydroxide.

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This is also beneficial for LoC-SERS bioanalysis applying the in situ nanoparticle synthesis. Nitrogen formed in the reaction remained dissolved in water since no gas bubbles were

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observed in the channels of the microfluidic chip by visual inspection. The pH value of the final colloidal solution is found to be 9.5.

The in situ colloid synthesis was performed in a segmented flow based microfluidic platform (Figure 1) by pumping the solutions A (silver nitrate 2 mmol L-1, sodium citrate 0.8 mmol L-1

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and ammonium hydroxide 18 mmol L-1 ) and B (hydrazine hydrate 1 mmol L-1) into a stream of mineral oil. Batch synthesized nanoparticles were prepared by mixing the equal volumes of the solutions A and B.

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In Figure 2, a significant difference in the shape and size of the particles prepared in a batch compared to particles prepared in situ is illustrated by SEM and TEM images as well as UV-

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VIS extinction spectra. Batch prepared particles are mostly round in shape and polydisperse in size (Figures 2A and B), while particles prepared in situ contain large amounts of truncated triangular particles besides some amount of round particles (Figure 2C). This might be a consequence of the different mixing profile in the chip compared to the mixing of the solutions in the large batch and/or a result of the impact of oil on the nanoparticle surfaces at the interface between water and oil, where the oil may play a role of a surfactant. To confirm the shape and size of the in situ prepared metallic nanoparticles present in the chip, PVP is applied to coat the nanoparticles directly in the chip conserving their structure for further 10

ACCEPTED MANUSCRIPT analysis. The TEM image of the particles coated with PVP (Figure 2D) indicates no significant structural change as compared with the particles collected in a chip without PVP. This observation serves as a clear indication that the nanoparticle formation is finished by the moment when the nanoparticles will be mixed with the analyte solution in the chip for SERS

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measurements, and no further change of the nanoparticle shape occurs. The UV Vis spectra depicted in Figure 2E indicate a shift of the localized surface plasmon (LSP) peak toward higher wavelengths in the case of in situ prepared colloids. This is probably associated with a

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change in the shape. The UV-VIS spectra of the in situ prepared particles coated with PVP have an almost identical maximum of the LSP peak, again illustrating no significant

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difference between in situ fabricated particles coated with PVP and those without a PVP layer. The SERS activity of the hydrazine reduced silver colloids in ammonium hydroxide solution was tested with several different analytes (Figure S1), showing the applicability of these colloids in SERS analysis of vast variety of analytes ranging from typically good SERS

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reporters (nile blue and 4-mercaptobenzoic acid), through biologically relevant analytes

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(thiamine and uric acid) to medications (levofloxacin).

Figure 2. A) SEM image of the nanoparticles produces in a large batch; B) TEM image of nanoparticles produced in a large batch; C) TEM images of nanoparticles produced in situ and D) TEM image of the PVP

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ACCEPTED MANUSCRIPT coated nanoparticles produced in situ. E) Absorption spectra of the a) nanoparticles produced in large batch, b) nanoparticles produced in situ and c) PVP coated nanoparticles produced in situ. Absorbance values were normalized to ease the comparison.

To test the SERS performance of the synthesized nanoparticles in a microfluidic setup

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adenine was chosen as a model analyte, due to its good SERS response.[33, 34] Figure 3 shows the Raman and SERS spectrum of the adenine molecule. The SERS spectrum given presents a cuvette-based SERS spectrum of a 1 µmol L-1 adenine solution in water. The most prominent

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band around 730 cm-1 corresponds to the ring breathing mode of the adenine and was applied for the integration of the peak area in further investigations. Observed differences in the

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relative peak intensities between Raman and SERS spectra of adenine are a consequence of the surface selection rules where Raman modes perpendicular to the metallic surface are preferentially enhanced.[35-37] Slight shifts of the peak positions in the SERS spectrum compared to the Raman spectrum of the adenine are the consequence of the change of the polarizability of the molecule as a result of the chemical binding of the adenine molecule to

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the silver surface.[38, 39] SERS spectra of adenine solutions of various concentrations were

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processed to calculate the integrated peak area of the ring breathing mode at 730 cm-1 (Figure

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S2).

Figure 3. Illustration of a cuvette-based SERS spectrum of a 1 µmolL-1 adenine solution in water and a Raman spectrum of solid adenine.

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ACCEPTED MANUSCRIPT In a next step, batch prepared nanoparticles were applied as SERS substrate within the microfluidic system at two consecutive days. The recorded adenine SERS spectra for various concentrations were processed to calculate the integrated peak area of the ring breathing mode at 730 cm-1 (Figure S3). In Figure 4A, the integrated peak area is plotted as function of the

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concentration. The day-to-day comparison of the LoC-SERS concentration profiles of adenine with batch colloids indicates good agreement between both sets of data for low

concentrations; however, the data analysis illustrates major differences in the SERS activity

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for higher concentrations. The different saturation behavior might be related to a varied

amount of free binding sites on the metallic surface. More likely, the aging of the colloid

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results in pre-aggregated clusters which yield in a higher SERS intensity. However, this observation is not reproducible, which is a drawback of batch produced colloids. In Figure 4B, the linear range between 0.1 and 1.0 µmol L-1 is highlighted. Based on the LOQ (Limit of quantification) values estimated on both measuring days (Table 1), a quantitative

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measurement is only feasible in a narrow working range, i.e. between 0.45 and 1 µmol L-1.

Figure 4. A) Reproducibility of the LoC-SERS adenine measurements with colloids prepared in a large batch as function of the measured concentrations. B) Linear range of the LoC-SERS adenine concentration measurements with colloids prepared in a large batch.

As illustrated in Figure 5A, employing in situ prepared colloids, the reproducibility is improved significantly for all measured concentrations at two consecutive days (the 13

ACCEPTED MANUSCRIPT corresponding SERS spectra are depicted in Figure S3). Figure 5B shows, that linearity is observed in the range between 0.1 µmol L-1 and 2.5 µmol L-1. Based on the calculated LOD (Limit of detection) and LOQ values summarized in Table 1, quantification is improved down to 0.1 µmol L-1. Therefore, it is clearly demonstrated that in situ produced nanoparticles

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show a better performance, allowing for lower detection and quantification limits as well as a wider working range as compared to the nanoparticles synthesized in a large batch. This can also be correlated with the observation that in situ synthesized nanoparticles show roughly

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one order of magnitude higher SERS enhancement compared to the batch produced colloids

are given in Tables S1 and S2.

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(Figure S4). The mean values of integrated peak areas and corresponding standard deviations

Table 1. Calculated LOD and LOQ values for the LoC-SERS adenine concentration measurements with batch produced and in situ produced colloids.

LOQ

(µmol L-1)

(µmol L-1)

Day 1

0.145

Day 2

0.209

Day 1 Chip

0.448

0.0336

0.0593

0.0327

0.0495

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Day 2

0.431

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Batch

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LOD

Since long term reproducibility is essential for quantitative SERS based measurements, the experiment was conducted to determine the reproducibility of the measurement with in situ produced nanoparticles in the time frame of two months. The same measurement conditions were employed, whereas, new batch of the chemicals were used for the preparation of the solutions A and B. The concentration-dependent LoC-SERS intensity profile of adenine measured after two months with new A and B solutions (Figure 5) shows a profound 14

ACCEPTED MANUSCRIPT agreement with the previously obtained data. This relieves us of the limitations regarding colloid stability, since each prepared micro-batch in a chip yields the same performance. Since reproducibility was unaffected by changing the chemicals used for the synthesis, a certain robustness of the synthesis is imminent. No data normalization was employed since

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the used Raman setup provides stable measurement conditions over time (Figure S5).

Figure 5. A) Reproducibility of the LoC-SERS adenine measurements with colloids prepared in situ as function

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of the measured concentrations. B) Linear range of the LoC-SERS adenine concentration measurements with

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colloids prepared in situ.

Even though the proposed in situ nanoparticle synthesis was shown to be highly reproducible,

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we aimed to increase the working range. To realize this, an experiment was performed in which increased concentrations of the reactants were used for the preparation of the solutions

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A and B, i.e. solution A contained 4 mmol L-1 silver nitrate, 1.6 mmol L-1 sodium citrate and 22 mmol L-1 ammonium hydroxide, while solution B contained 2 mmol L-1 hydrazine hydrate. The analogue LoC-SERS measurements were performed with colloids prepared in situ employing the same flowrates and measurement conditions as in the previous experiment. As shown in Figure 6, with an increase of the silver amount, the linear working range was widened up to 6 µmol L-1 due to the formation of a larger number of the particles, which resulted in an increase of the effective surface of the particles available for adsorption of adenine. 15

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Figure 6. LoC-SERS adenine concentration profile with colloids prepared in situ applying increased reactant

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concentrations.

4. Conclusion

Silver reduction with hydrazine in ammonium hydroxide solution was successfully applied for

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the in situ colloid synthesis in a microfluidic platform utilizing glass microfluidic chip,

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directly prior to the SERS measurement. This strategy was shown to have an advantage over conventional synthesis in large batches. Since each droplet contains a freshly prepared batch

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of colloids, together with well-defined temporal control offered by the microfluidic setup and taking the short time between colloid formation and LoC-SERS measurement into account,

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the aging of the colloids presents no issue for the in situ colloid synthesis for LoC-SERS measurements. The colloids produced in the microfluidic setup are used for SERS measurements in situ shortly after formation, and with always identical time passing from the point of the colloid formation to the point of LoC-SERS measurement. Therefore, the colloids produced in situ do not need to meet the requirement of a long shelf life. Furthermore, higher enhancement is observed in SERS measurements performed with in situ prepared colloids, as the SERS signal is one order of the magnitude higher compared to the measurements with batch produced colloids. As a result, lower detection limits and wider working ranges were 16

ACCEPTED MANUSCRIPT obtained. Moreover, the working range can be further widened if needed, by the employment of a higher concentration of silver in the synthesis, which is not the case with an already prepared batch of the colloids. It is demonstrated that the measurements are highly reproducible in day to day comparison as well as in long term comparison. The great

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advantage offered by in situ colloid synthesis in a microfluidic chip combined with the

already proven potential of the LoC-SERS technique, raises the possibility for the application of this concept in vast variety of applications, ranging from the environmental protection,

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food safety, quality control to bioanalytics. With a low consumption of the reactants for the nanoparticle synthesis as well as low amount of the sample needed for the analysis, LoC-

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SERS with in situ colloid synthesis may become a method of choice for the point-of-care analysis and on-line monitoring applications, i.e. pollution control. It features a simple and reproducible way of the measurements with high sensitivity. Furthermore it offers the possibility of automatization, allowing personnel without special training to run the entire

Acknowledgements

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procedure of the nanoparticle synthesis and SERS measurements.

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Funding the PhD project of V. Dugandžić by “Carl-Zeiss-Strukturmaßnahme” is gratefully acknowledged. The projects “InfectoGnostics” [grant number 13GW0096F] and “Jenaer

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Biochip Initiative 2.0” [grant number 03IPT513Y] within the framework “InnoProfile Transfer – Unternehmen Region“ are supported by the Federal Ministry of Education and Research, Germany (BMBF). We thank the microfluidic group of the IPHT for preparing the lab-on-a-chip devices for the measurements. TEM and SEM images were acquired at the facilities of the Jena Cener for Soft Matter (established by grants of the DFG and EFRE). Additionally, the help of Martin Jahn with the data processing is gratefully acknowledged.

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ACCEPTED MANUSCRIPT [10] A. Kamińska, E. Witkowska, K. Winkler, I. Dzięcielewski, J.L. Weyher, J. Waluk, Detection of Hepatitis B virus antigen from human blood: SERS immunoassay in a microfluidic system, Biosensors and Bioelectronics, 66 (2015) 461-467. [11] Z. Wu, Y. Liu, X. Zhou, A. Shen, J. Hu, A “turn-off” SERS-based detection platform for

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ultrasensitive detection of thrombin based on enzymatic assays, Biosensors and Bioelectronics, 44 (2013) 10-15.

[12] J.H. Granger, M.C. Granger, M.A. Firpo, S.J. Mulvihill, M.D. Porter, Toward

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development of a surface enhanced Raman scattering (SERS) based cancer diagnostic immunoassay panel, The Analyst, 138 (2013) 10.1039/c1032an36128k.

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[13] K. Gracie, D. Lindsay, D. Graham, K. Faulds, Bacterial meningitis pathogens identified in clinical samples using a SERS DNA detection assay, Analytical Methods, 7 (2015) 12691272.

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applications, and the future, Materials Today, 15 (2012) 16-25.

[15] K. Faulds, A. Hernandez-Santana, W.E. Smith, The inorganic chemistry of surface enhanced Raman scattering (SERS), Spectroscopic Properties of Inorganic and

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Organometallic Compounds: Techniques, Materials and Applications, Volume 41, The Royal Society of Chemistry2010, pp. 1-21.

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[16] J.M. McLellan, Z.-Y. Li, A.R. Siekkinen, Y. Xia, The SERS Activity of a Supported Ag Nanocube Strongly Depends on Its Orientation Relative to Laser Polarization, Nano Letters, 7 (2007) 1013-1017.

[17] E. Nalbant Esenturk, A.R. Hight Walker, Surface-enhanced Raman scattering spectroscopy via gold nanostars, Journal of Raman Spectroscopy, 40 (2009) 86-91. [18] K.G. Stamplecoskie, J.C. Scaiano, V.S. Tiwari, H. Anis, Optimal Size of Silver Nanoparticles for Surface-Enhanced Raman Spectroscopy, The Journal of Physical Chemistry C, 115 (2011) 1403-1409. 19

ACCEPTED MANUSCRIPT [19] Y. Zhang, B. Walkenfort, J.H. Yoon, S. Schlucker, W. Xie, Gold and silver nanoparticle monomers are non-SERS-active: a negative experimental study with silica-encapsulated Raman-reporter-coated metal colloids, Physical Chemistry Chemical Physics, 17 (2015) 21120-21126.

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[20] W. Shen, X. Lin, C. Jiang, C. Li, H. Lin, J. Huang, S. Wang, G. Liu, X. Yan, Q. Zhong, B. Ren, Reliable Quantitative SERS Analysis Facilitated by Core–Shell Nanoparticles with Embedded Internal Standards, Angewandte Chemie, 127 (2015) 7416-7420.

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[21] S. Lee, J. Choi, L. Chen, B. Park, J.B. Kyong, G.H. Seong, J. Choo, Y. Lee, K.-H. Shin, E.K. Lee, S.-W. Joo, K.-H. Lee, Fast and sensitive trace analysis of malachite green using a

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surface-enhanced Raman microfluidic sensor, Analytica Chimica Acta, 590 (2007) 139-144. [22] A. Marz, T. Henkel, D. Cialla, M. Schmitt, J. Popp, Droplet formation via flow-through microdevices in Raman and surface enhanced Raman spectroscopy-concepts and applications, Lab on a Chip, 11 (2011) 3584-3592.

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[23] E. Kammer, K. Olschewski, T. Bocklitz, P. Rosch, K. Weber, D. Cialla, J. Popp, A new calibration concept for a reproducible quantitative detection based on SERS measurements in a microfluidic device demonstrated on the model analyte adenine, Physical Chemistry

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ACCEPTED MANUSCRIPT Silver Nitrate with Hydroxylamine Hydrochloride, The Journal of Physical Chemistry B, 107 (2003) 5723-5727. [27] R. Blue, D. Uttamchandani, Recent advances in optical fiber devices for microfluidics integration, Journal of Biophotonics, 9 (2016) 13-25.

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ACCEPTED MANUSCRIPT [36] L. Xia, M. Chen, X. Zhao, Z. Zhang, J. Xia, H. Xu, M. Sun, Visualized method of chemical enhancement mechanism on SERS and TERS, Journal of Raman Spectroscopy, 45 (2014) 533-540. [37] A. Campion, J.E. Ivanecky, C.M. Child, M. Foster, On the Mechanism of Chemical

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Highlights

In situ synthesis of silver colloids for SERS in a microfluidic chip

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Reduction of Ag+ by a hydrazine in ammonium hydroxide solution

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Better day-to-day and long term reproducibility compared to batch synthesized colloids

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Possibility to widen the working range.

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