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Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device
Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device K. Karthikeyan* and L. Sujatha Abstract— Mercury (Hg2+) ion is one of the heavy metal ions present in water and highly poisonous to human consumption. Thus, developing an efficient sensor for detection of mercury ions at different concentration levels in the water down to nM is essential. Here, we report the development of fluorometric sensor using the Fluidic MEMS Device (i.e., microfluidic device) with high sensitivity and selective detection of Hg2+ in water using tiny quantity of sensing fluids (~2.8 μL). L-Arginine capped gold nanoparticles functionalized with Rhodamine 6G allowed the onchip fluorescence detection of Hg2+ ions concentration regime from 0 to 16 nM in water samples. The developed microfluidic device senses the presence of Hg2+ ions through the change in intensity of fluid fluorescence. The sensor response is found to be linear with respect to the Hg2+ ions concentration ranging from 2 to 12 nM. Index Terms—Microfluidic Device, Fluidic MEMS Device, Gold Nanofluid, Mercury Detection, Fluorometric
I. INTRODUCTION The presence of mercury (Hg2+) in water is one of the significant hazardous for human health and environment. This metal ions cause several diseases like cardiac disorders, neurological, and several developmental illnesses. Most of the surface and groundwater gets polluted by untreated sewage water, industrial wastage, gasoline, batteries wastage, poor sanitation management, pigments, medical waste, electroplated steel and electronics spares. It is essential to monitor the presence of Hg2+ in drinking water system by low cost, high-sensitive and selective detection methods [1-3]. Several techniques have been used for the detection of Hg2+ present in water. The techniques for detection includes highresolution surface plasmonic resonance spectroscopy [4], atomic absorption spectrometry [5], surface-enhanced Raman spectroscopy [6], inductively coupled plasma atomic emission spectrometry [7], fluorescence microscope [8], colorimetric [9], and electrochemical [10- 13]. The problems with conventional techniques includes expensive instruments, highly skilled labors, huge execution K Karthikeyan, Research Associate, Centre of Excellence in MEMS & Microfluidics, Rajalakshmi Engineering College, Thandalam, Chennai, India. Mobile No: +91-9944477213; E-mail:
[email protected] L Sujatha, Professor & Head, Centre of Excellence in MEMS & Microfluidics, Rajalakshmi Engineering College, Thandalam, Chennai, India. Mobile No: +91-9444847007; E-mail:
[email protected]
time, costly reagents and a large volume of reagent samples. On the other hand, microfluidic device based sensor is a promising alternative to the conventional method. It allows to achieve complete laboratory protocols on a single chip of few square centimeters. Moreover, a microfluidic device is an emerging technology in handling chemicals and reagents in the range of micro and nanoliters. This technology helps in improving the performance of the device in terms of rapid mixing and reaction process. The miniature microfluidic devices were used in different bio analysis such as micro polymerase chain reaction (µPCR) for salmonella detection [14], synthesis of nanoparticles and biomaterials [15], detection of hormonal compounds [16], cell sorting [17], detection of foodborne pathogens [18], detecting and differentiating tumour cells [19]. The microfluidic based heavy metal ion detection such as detection of lead in y type microchannel by fluorescence method and Calix-DANS4 as a sensing material [20], deoxyribozyme immobilization on PMMA based microfluidic device walls for the detection of Pb2+ [21] and a zigzag microchannel designed for the detection of Pb2+ (10μM) by chelation mechanism using 11mercaptoundecanoic acid (MUA)-functionalized gold nanoparticles (MUA-AuNPs) as a sensing probe [22] were reported. A microfluidic chip for the detection of cobalt (II) in water using chemiluminescence method with the detection limit of 5.6*10-11 mol/L [23] was presented. An electro chemiluminescence method on a lab chip to detect Hg2+ ions in water using amorphous thin film diodes was explained and the detection range was 66 nM–100 nM [24]. The materials such as bismuth, gold, silver based particles possess significant photocatalytic activity which have been used for heavy metal ion detection [25-27]. Several research articles report that heavy metal mercury ion detection using gold nanoparticles has excellent optical, physical and chemical properties. Colorimetric based sensor using 11mercaptoundecanoic acid (MUA) -capped gold nanoparticles with amino acids were reported to detect Pb2+ heavy metal ions in the range between 2 and 50 μM [28-31]. A digital microfluidic device based colorimetric sensor was fabricated to detect mercury ions [32]. The complex of mercury-specific oligonucleotide materials and poly (3-(3 -N, N, N-triethylamino-1-pro-pyloxy)-4-methyl-2,5-thiophene hydro-chloride) (PMNT) was used as a sensing probe for mercury ion detection. The color changes for various concentration levels of mercury ions were demonstrated and
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Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device the detection limit was 6.25 - 100 μM with 2µL volume of sensing fluid. But this sensitivity level is less compared with our proposed sensing probe and method. A colorimetric based mercury ion detection was developed using L-cysteine functionalized gold nanorods [33]. The developed sensor has a lower detection limit as 3 ppt which is very low but with the usage of 3mL of sensing fluid. This volume is very large compared to our proposed technique with better sensitivity as suggested by WHO. Several herringbone type micromixers were reported for better mixing process and those structures are highly complex design to fabricate, more pressure drop during fluid flow and also complete mixing achieved at high flow rates [34-38]. This high flow rate will damage the device bonding and will affect the life time of the microfluidic device. However, the sensitivity and selectivity for metal ion detection using gold nanoparticles remain challenging. Rhodamine 6G is an excellent fluorescence material which can be adsorbed on gold nanoparticles and the fluorescence emission signal of gold nanoparticle surface attributed to the localized plasmonic field was explained [39]. Amino acids can bind with gold nanoparticles and it can form complexes with heavy metal ions through their amino groups. Detection of heavy metal mercury ions in water using microfluidic device using bovine serum albumin (BSA) - rhodamine 6G (R6G) – gold nanoparticles and gold nanoclusters as sensing probes using a digital camera as the detector was reported [39] and the detection limit is 0.6 μg/L at the centre of the flowershaped meandering channel with 180 mm mixing length and 5 µl volume of fluid. But our designed micromixer is a very simple structure so that pressure drop is less and mixing efficiency is high at a low flow rate. In our proposed design, the micromixer length is 95 mm and the volume of fluid is approximately 2.8 μL. These length and volume of sensing fluids are very less compared to the previously reported methods. In this work, we are choosing L-Arginine amino acid functionalized with gold nanofluids and rhodamine 6G for high sensitivity and selective detection of Hg2+ fluorescence based detection. Hence, Rhodamine 6G and L-Arginine would be suitable materials to modify gold nanoparticle surface for selective detection of the Hg2+ ion. Also, we proposed to fabricate an inexpensive fluorometric microfluidic device for detection of heavy metal Hg2+ ions with new and simple herringbone type micromixer device. This structure reduces the volume of sensing fluids (μL) and provides better mixing performance. The microfluidic device was fabricated on Poly Dimethyl Siloxane (PDMS) using soft lithography technique. The combination of L-Arginine, rhodamine 6G and gold nanofluids in microliter quantity was used as sensing probe and it is one of the inlet fluid for detection of toxic mercury ions. The known concentration of heavy metal ions in aqueous media is used as other inlet fluid. II. EXPERIMENTAL
The herringbone (HB) type micromixer device has two inlets such as inlet A and B, with each inlet port has a diameter of 3 mm. The inlet channels are at an angle of 45 degrees with the x-axis, and the lengths are of 3.25 mm. The herringbone microchannel is the mixing zone whose length is 95 mm with eight bends on each side and the diameter of the sensing zone is 5 mm. The width of the microchannel is 200 µm and spacing between adjacent microchannel is 500 µm. The outlet port diameter is also 3 mm similar to the inlet port. The total chip length is 26 mm in x-axis and chip width is 13.5 mm in the y-axis. Fig.1 shows the design of y shape herringbone type micromixer.
Fig. 1. Design of Y Shape Herringbone Type Micromixer
B. Fabrication Process of Microfluidic Device In soft lithography technique, a rigid stamp (prime mould) with the pattern is used to generate replica mould patterns (castings). In our experiments, the prime mould was fabricated using UV lithography technique on SU-8 2075 (MicroChem, USA) negative photoresist. It was spin coated onto a silicon substrate at a spin speed of 2200 rpm for 30 seconds to achieve 100µm thickness. The sample was soft baked for 5 minutes at 65 ˚C then for 15 minutes at 95 ˚C. SU-8 was exposed to UV exposure system (365 nm wavelength) for 12 seconds. Then the sample was post-baked for 10 minutes at 95 ˚C. Then, it was developed using SU-8 developer solution in the ultrasonicator bath for 12 seconds. Now, with the desired HB pattern on SU8, the sample was hard baked for 1 hour at 70˚C. The fabricated SU-8 prime mould structure was kept in a petri dish. Afterwards, PDMS prepolymer was prepared with silicone elastomer and the curing agent (Sylgard 184, Dow Corning, USA) as 10:1 ratio and mixed well. To eliminate air bubbles, the mixture was kept in vacuum desiccators for 1 hr. Then it was poured on the SU-8 prime mould structure and cured at 70ºC for 3 hrs. After cooling down to room temperature, the PDMS layer was peeled off from the SU-8 pattern. The follow-on PDMS micro mould structure is a replica structure of SU-8 prime mould structure. We could use this prime mould for multiple times to fabricate PDMS micro mould. The PDMS-based channel was finally bonded with glass cover plate using oxygen plasma treatment. Fig.2 shows the fabricated HB micromixer with two inlets and an outlet. The top side of the device is made up of PDMS and the bottom side is a glass cover plate.
A. Design of Device Structure
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Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device Inlet fluid B is an analyte which contains a known concentration of mercury metal ion in an aqueous medium. Analytes were prepared for known concentrations of 0, 2, 4, 6, 8, 10, 12, 14 and 16 nM of Hg2+ in DI water. E. Experimental procedure of metal ion detection process
Fig. 2. Photograph of fabricated HB micromixer with inlets and outlet
C. Preparation of gold nanofluids Gold nanofluids (AuNFs) were prepared using sodium citrate reduction method. 50ml of 1mM HAuCl4.3H2O (SRL, India) was taken in a beaker and kept on a magnetic stirring hot plate at 80°C for 20-30 minutes. 2ml of 1% trisodium citrate (SRL, India) was added to the above hot solution. Then the resulting mixture was cooled down to room temperature and the obtained AuNFs was kept at 4°C in a dark place [40]. Fig.3 shows the surface morphology of AuNFs studied using the high-resolution transmission electron microscope (HRTEM) - (a) Spherical shape gold nanoparticles (b) The measured diameter of the particle size is 18 nm and (c) a spacing of fringe is 0.20 nm.
For mercury detection, fluids are simultaneously entering at inlet ports A and B and were mixed while they pass through the herringbone type micromixer. Both the fluids were mixed well in the mixing zone and collected at the sensing zone where the fluorescence study is being carried out. Then the fluid exits through the outlet port of the device. For fluorescence response, the sensing fluid at sensing zone of the device was exited at 350nm. Then the fluorescence response was read using portable fluorescence spectrometer. The fluorescence study shows variation in the intensity with respect to the presence of Hg2+ ions. Fig.4 shows the graphical working procedure for the detection process.
Fig. 4. Graphical working procedure for detection process
III. RESULTS AND DISCUSSION A. FTIR Studies of Sensing fluids (a)
(b)
(c) Fig. 3. HRTEM images of Gold nanoparticles (a) Low magnification (b) High magnification (c) spacing of fringe is 0.20 nm
The prepared AuNFs are functionalized with L-arginine and Rhodamine 6G. The procedure for the function is as follows: 0.2 mg of L-arginine and 0.5mM of Rhodamine 6G were taken in a vial containing AuNFs and stirred at room temperature for 15 minutes. The obtained sensing probe (LArg-GN-R) will be used as inlet fluid A. D. Preparation of Sample Fluid
The chemical interactions between gold nanoparticles (AuNPs), L-Arginine and Rhodamine 6G were done using FTIR in the range of 400 cm-1 - 4000 cm-1. Fig. 5. (a) shows the FTIR spectrum of pure AuNPs. The peaks are assigned to C-H, C = O and O-H chemical bonds which are derivative from sodium citrate. The AuNPs spectrum is achieved in the lines attributed to C-H bonds at 2942 cm-1, C = O bonds at 1630 cm-1 and O-H bonds at 3152 cm-1. Fig. 5 (b) shows the FTIR spectrum of AuNPs capped by L-Arginine. The assigned peaks in the region from 3638 cm-1 to 3853 cm-1 are due to OH stretching absorption of water and COOH molecules existing in the sample. Peak at 2355 cm-1 is due to N-H stretching vibration spectra in L-Arginine capped AuNPs. The assigned peak at 1546 cm-1 is due to primary amine group N-H bending vibration. The peak at 1450 cm-1 is due to asymmetric deformation vibration in CH3. The peaks at 1140 cm-1 indicate symmetric C-N stretching band. The band at 676 cm-1 is due to L-arginine capped AuNPs assigned due to COO- plane deformation. It is observed that a significant shift in COO- and NH3+ stretching are likely due to the change in dipole moment when L-arginine binds to AuNPs surface. The
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Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device characteristic peaks in the FTIR spectra show the strong bonding between AuNPs and L-Arginine functional groups.
decreases. The device shows a linear behavior for the concentrations upto 12 nM with the minimum detection limit as 2 nM.
Fig. 6. Fluorescent intensity of different concentrations of Hg2+ Vs Wavelength (nM) Fig. 5. FT-IR spectrum of (a) AuNPs (b) AuNPs capped by L-Arginine and (c) AuNPs capped by L-Arginine & Rhodamine 6G
Fig. 5. (c) shows the FTIR spectrum of AuNPs capped by LArginine and Rhodamine 6G. The assigned peaks in the region at 661 cm-1 and 693 cm-1 are due to C–C–C ring in plane bending. The peaks at 794 cm-1,980 cm-1 and 1082 cm-1 are due to C–H out plane bending. The peaks at 1138 cm-1 are due to C-H in plane bending. The peaks at 1241cm-1 and 1432 cm-1 correspond to C–O–C stretching and C–N stretching respectively. The peaks at 1524 cm-1 and 1635 cm-1 are confirming aromatic C–C stretching. The results match with the reports in the literature [41-42]. B. Fluorescence studies For mercury detection, Fig.6 shows the fluorescent intensity spectrum of L-Arg-GN-R with respect to Hg2+ concentrations ranging from 0-16 nM (bottom to top) Vs Wavelength (nM). At 0 nM of Hg2+, there is no visible peak in the spectra. When the concentration level of Hg2+ increases from 2 – 16 nM the fluorescence intensity increases. The maximum fluorescent intensity level 532 was achieved at 16 nM with a phenomenal change in the fluorescence emission intensity. This fluorescent enhancement is credited to the formation of the L-Arg-GN-RHg2+ complex. Fig.7 shows the error rate in fluorescent emission intensity due to complex formation of L-Arg-GN-RHg2+ Vs at different concentration level (0-16 nM). We found that the L-Arg-GN-R-Hg2+ complex can sense the metal ion concentration up to 16 nM of Hg2+ concentration. Beyond 12 nM concentration, the increase in fluorescent intensity
Fig. 7. Error rate in fluorescent intensity of Hg2+ detection Vs Different Concentration Levels (0,2,4,6,8,10,12,14 & 16 nM)
Fig.8 shows the selectivity in fluorescent intensity for different metal ions in water at 16 nM concentration with the applied flow rate of 1μL/min. This selectivity study was carried out with different metal ions separately and L-Arg-GN-R as sensing fluid. These fluids are allowed to flow into the designed micromixer device and the measured fluorescence emission intensity was studied. The experiments were carried out by determining the changes in the fluorescent emission intensity by the addition of metal ions such as Hg2+ Al3+, Ba2+, Cu2+, Cr2+, Co2+, Mn2+, Mg2+, Pb2+ and Zn2+ to the solutions of L-Arg-GN-R.
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Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device
TABLE I Comparison of mercury ion detection of developed microfluidic device Sensing Materials Detection Detection Method Detection Metal ions Range Gold nanoparticles with porous Hg2+ Surface enhanced 1 to 20 ppb anodic alumina Raman spectroscopy Gold electrode Hg2+ Surface plasmon 2 ppb resonance with anodic stripping voltammetry Agar powder modified with 2 Hg2+ Cold vapor atomic 0.040–2.40 mercaptobenzimidazole and absorption spectrometry ngmL−1 SnCl2 Aminodibenzo-18-crown-6 Hg2+ Surface enhanced 1 x 10-11 M coupled with Raman spectroscopy to mercaptopropionic acid and the 1 x 10-6 M resultant crown ether derivative Thiourea and gold(III) chloride Hg2+ Inductively coupled 0.1-2.0mgL1 plasma with atomic emission spectroscopy 1-[2-pyridylazo]-2-naphthol Hg2+ Spectrophotometric 1.0 to on triacetyl cellulose 1000.0 μM Gold - dimethyl amino ethane Hg2+ Square wave anodic 20 ppb to thiol - single walled carbon stripping voltammetry 250 ppb nanotube-poly (m-amino benzene sulfonic acid) 1,10-phenanthroline and its Hg2+, Pb2+ Screen Printed 50 μM – derivative naphtho[2,3Phenanthroline based 1mM a]dipyrido [3,2-h:2',3'Flexible Electrochemical f]phenazine-5,18-dione Trisphenanthroline Hg2+ Electro60 nM ruthenium(II) complex chemiluminescence Mercaptopropionic acid Hg2+, Pb2+, Fluorescent spectra 9.6x10-8 to 2+ modified Au NPs and 2,6Cu using 6.4x10-6 M, pyridinedicarboxylic using microfluidic acid device 11-mercaptoundecanoic acidHg2+, Cd2+, Colorimetric method 2-50 μM capped gold Fe3+, Pb2+, nanoparticles Al3+, Cu2+, Cr3+ poly (3-(3’-N,N,NHg2+ Colorimetric method 6.25 – 5000 triethylamino-1’-pro-pyloxy)-4using digital μM methyl-2,5-thiophene microfluidic device hydrochloride) (PMNT) and This area of 12mm × mercury-specific 6mm oligonucleotide (MSO) probe Bovine serum albumin - gold Hg2+ Fluorescent spectra 0.6 μg L-1 nanocluster using microfluidic 15 μg L-1 device – 100 μm of deep, 300 μm wide and 180 mm long L-Arginine capped gold Hg2+ Fluorescent spectra 2 - 12 nM nanoparticles functionalized using Herringbone shape with Rhodamine 6G microfluidic device – 100 μm of depth, 200 μm of width and 95 mm
References [3] [4]
[5]
[6]
[7]
[9] [10]
[13]
[24] [26]
[28]
[29]
[36]
Proposed detection
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Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device
From this study, it was observed that Pb2+ ions promote small fluorescence intensity changes compared with other metal ions. But this intensity change is very less compared with Hg2+. Thus, the fluorescent emission intensity observed for Hg2+ compared with other metal ions is the highest selectivity of L-Arg-GN-R. Fig 8 also shows the error rates and it denotes ±12% uncertainty in the fluorescent spectrum of different metal ions in water.
that the ion-exchange of the analyte is higher at lower flow rates, as well as the mixing with sensing solution, which promotes the detection. While at higher flow rates, there is a decrease of fluorescence response due to insufficient time for mixing and the higher flow rate may also cause severe damage to the channel by its high pressure. Based on the experimental results, we propose the sensing mechanism which is based on the high-affinity metallophilic interactions of Hg2+, which results in an increase of the fluorescence intensity of L-ArgGN-R. The device dimensions and sensitivity of our proposed sensor has been compared with other reported Hg2+ sensors and listed in Table 1. IV. CONCLUSION The microfluidic device with a herringbone structure was successfully designed and fabricated for the detection of mercury ions in water. The fluorescent sensing probe L-ArgGN-R was synthesized, characterized and applied for the detection of Hg2+. L-Arg-GN-R shows a linear response for concentrations in the range of 2-12nM. The device shows excellent selectivity towards Hg2+ when compared to other ions tested under the same conditions. Highly sensitive fluorescence detection was realized by the fluorometricmicrofluidic device for the detection of Hg2+ ions with the herringbone microchannel structure.
Fig. 8. Selectivity & Error rates in Fluorescent intensity of different metal ions in water (16 nM concentration)
Fig. 9. Error rates - Fluorescent intensity of Hg2+ Vs Different flow rates (1,25,50,75,100,125,150,175& 200 µL/min)
Fig.9 shows the fluorescence intensity of Hg2+ ion mixer solution containing 16nM with different flow rates 1, 25, 50, 75, 100, 125, 150, 175 and 200 µL/min. The results indicate that the flow rate has a significant influence on the fluorescence signal. From the fluorescence study, it is inferred
ACKNOWLEDGMENTS We acknowledge Defense Research and Development Organization (DRDO) for the financial support under Extramural Research and Intellectual Property Rights (ER&IPR) for the fabrication of microfluidic devices using soft lithography.
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This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/JSEN.2018.2840331, IEEE Sensors Journal
Manuscript No. Sensors-21337-2018, Fluorometric Sensor for Mercury Ion Detection in a Fluidic MEMS Device
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K Karthikeyan, B.E., M.Tech., [Ph.D.] - is a Research Associate in Centre of xcellence in MEMS & Microfluidics, Department of Electronics and Communication Engineering, Rajalakshmi Engineering College, Chennai, India. He is currently pursuing the Ph.D. degree in technology at Anna University, Chennai, Tamil Nadu, India. His research interests are Design, Fabrication and Packaging of Microfluidic Devices, MEMS Transducers and Nanomaterials
Dr L Sujatha, Head, Centre of Excellence in MEMS & Microfluidics (CEMM) and Professor in the Department of Electronics & Communication Engineering, Rajalakshmi Engineering College (REC), Chennai. She has 27 years of experience in teaching and research. She had done her research in the field of Micro Electro Mechanical Systems (MEMS) at Indian Institute of Technology Madras and received her Ph.D degree in 2008. She had also done her Post-Doctoral Fellowship at IIT Madras for the fabrication of an Optical Biosensor during 2011-12. She has published several papers in refereed international journals, more than 40 International Conferences and given invited talks at various Universities. She is handling a number of sponsored projects funded by AICTE, AERB, DRDO and DST. Her research interests are fabrication of MEMS and Micro-Fluidic Devices for chemical and biosensors. sensors.
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