microfluidics based handheld nanoparticle synthesizer · original paper microﬂuidics based...

ORIGINAL PAPER

Microfluidics based Handheld Nanoparticle Synthesizer

S. C. G. Kiruba Daniel1 • Lourdes Albina Nirupa Julius1 •

Sai Siva Gorthi1,2

Received: 15 July 2016 / Published online: 28 November 2016

� Springer Science+Business Media New York 2016

Abstract Current study relates to the development of an electrical power-free,

handheld microfluidic nanoparticle synthesizer for synthesis of uniform sized silver

nanoparticles at room temperature. The synthesizer module consists of a custom

designed microreactor and employs negative pressure based pumping mechanism

for the electrical power free synthesis of metal nanoparticles. In order to realize a

microreactor capable of on-site synthesis of monodisperse nanoparticles, opti-

mization studies by bulk biosynthesis at varying ratios of the precursor and the

reducing agent followed by UV–VIS absorption studies were performed to deter-

mine the appropriate mixing ratio. Later, a custom designed microfluidic micro-

mixer was used to perform volumetric flow rate optimizations at the desired ratio

using syringe pumps. From the knowledge of the precursor and reducing agent ratio

and the flow rates, we modified the hydraulic resistance of micro-mixer inlets by

varying the channel geometry to meet the optimized specifications leading to

effective synthesis. The synthesized nanoparticles were characterized by UV–VIS

spectroscopy, XPS, FTIR, EDS, HRTEM and SAED. The crystal lattice planes of

[111] and [220] from the SAED pattern confirms the presence of silver nanoparti-

cles. HRTEM study elucidates that the size of the synthesized nanoparticles is

between 2 and 10 nm.

S.C.G. Kiruba Daniel and Lourdes Albina Nirupa Julius have equally contributed.

Electronic supplementary material The online version of this article (doi:10.1007/s10876-016-1120-x)

contains supplementary material, which is available to authorized users.

& Sai Siva Gorthi

[email protected]; [email protected]

1 Shanmukha Innovations Pvt. Ltd, First Floor, SID Building, IISc, Bangalore 560012, India

2 Department of Instrumentation and Applied Physics, Indian Institute of Science (IISc),

Bangalore 560012, India

123

J Clust Sci (2017) 28:1201–1213

DOI 10.1007/s10876-016-1120-x

http://dx.doi.org/10.1007/s10876-016-1120-x

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Keywords Microfluidics � Nanoparticle synthesizer � Silver nanoparticles �Biosynthesis

Introduction

The functionalities of metal nanoparticles has revolutionized many areas of research

including drug delivery, diagnosis, sensing, water purification, effluent treatment,

electronics, solar cell, etc. [1–7]. Conventional techniques of metal nanoparticle

synthesis utilize microwaves, hydrothermal based methods, sol–gels, sonication, co-

precipitation and biosynthesis (plant/microbes) [8–13].

Most of the above mentioned techniques demand either maintenance of specific

ambient conditions or require generation of physical effects like stirring, shaking,

sonic waves, microwaves, etc. for the proper synthesis of nanoparticles. This limits

their use to laboratory environment. For instance, hydrothermal synthesis calls for

high pressure and temperature conditions. On the other hand, wet chemical

synthesis techniques like sol-gel, co-precipitation requires the usage of carcinogenic

or hazardous chemicals like sodium borohydride, sodium dodecyl sulphate (SDS),

etc., as strong/weak reducing agents, and further addition of stabilizing agents.

Biosynthesis, a method utilizing biogenic substances or plant based extracts as

reducing agents is an eco-friendly way of nanoparticle generation. They are

otherwise known as green synthesis. Room temperature biosynthesis methods have

been explored in literature. However, these batch-wise methods may result in highly

polydispersed nanoparticles [13].

Synthesizing nanoparticles with uniform size, shape, dispersity, surface area and

minimal environmental impact is crucial. With the advent of microfluidics, there is

enhanced controllability and reproducibility of properties of the nanoparticles.

Unlike off-chip bulk synthesis of nanoparticles, microfluidic on-chip synthesis

employs controlled flow rates of the precursor and reducing agent leading to

controlled size and shape of metal nanoparticles [14]. Microfluidic biosynthesis of

silver nanoparticles has been earlier reported by using tubular-fluidic system

[15, 16]. This microreactor comprises of tubular structure with size ranging from

few millimeters to sub-millimeter cross-section and extending up to few meters in

length. The reported biosynthesis protocol requires temperature control as they

utilize sundried leaf powder based extract. Extract prepared from the leaf powder

obtained after sun drying the leaves for a week results in possible loss of major

proteins and polyphenols, thus having a requirement of a heating mechanism which

was accomplished by immersing the tubular channel in a hot bath. Rapid room

temperature based microfluidic biosynthesis has not been reported as of now.

Another characteristic feature of microfluidics is the ability to transform complex

laboratory processes and systems to miniaturized ones as in Lab-on-a-Chip (LoC).

By capitalizing on this aspect we explored the possibility of developing a simple

cost effective system for controlled synthesis. This can be made available at remote

resource limited settings for a plethora of applications. Although many nanoparticle

synthesizers which are based on the principles of flame ionization, millifluidics, etc.

are currently available in the market, they are bulky and expensive. Hence there is a

1202 S. C. G. Kiruba Daniel et al.

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need for the development of a portable handheld nanoparticle synthesizer. To

address this need, the present study aims at the design and development of a

handheld, cost-effective in-situ nano-synthesizer for the synthesis of metal

nanoparticles.

Present work deals with the development of a green synthesis protocol for the

synthesis of silver nanoparticles at room temperature in a microfluidic platform. The

procedure does not involve the use of any hazardous chemicals and works well

without the need of external stirring or shaking. In-order to accomplish this, various

optimization studies were carried out. The synthesis was carried out in a

Polydimethylsiloxane (PDMS) based microfluidic reactor. The synthesis of silver

nanoparticles uses silver nitrate as the precursor and Parthenium leaf extract as the

reducing agent. Previously silver nanoparticles synthesized off-chip (batch process)

using Parthenium leaf extract [17] led to highly polydispersed nanoparticles

whereas in the current method involving microfluidics, monodisperse silver

nanoparticles were obtained by controlling the flow rates and channel sizes. The

microfluidic device design fabricated for the study has a planar serpentine micro-

mixer geometry that helps in effective mixing of the leaf-extract and the precursor

within a very small foot-space. The dimensions of the mixer are of few hundred

microns cross-sectional dimensions and few millimeters of length, including the

micro-mixing zone. This not only minimizes the device foot print but also reduces

the quantity of reagents used. The usage of very less precursor and reducing agent

(in microliters) to produce nanoparticles proves useful for applications requiring

less sample volume like sensing and bio barcoding. Also, the time taken for the on-

chip synthesis is significantly less (around 100 ms) compared to off-chip batch

synthesis. Finally, using electric circuit analogy a LoC design augmented with

negative pressure based pumping mechanism that facilitates the realization of a

handheld nanoparticle synthesizer is presented.

Materials and Methods

Preparation of Leaf Extract and Initial Off-Chip Biosynthesis ProtocolOptimization

Leaf extract has been prepared by boiling 30 g of leaves of Parthenium

histerophorus weed in 100 ml deionized water for 1 h at 373 K. Extract is filtered

through Whatman Paper and kept at 4 �C for storage. The prepared leaf extract can

be stored and used for 6 months (and does not require heating/cooling again at the

time of nanoparticle synthesis). The pH of the leaf extract is adjusted to 10 by the

addition of 0.1 N NaOH to it before synthesis. To get the optimum ratio of the

reducing agent to the precursor for the synthesis of silver nanoparticles several trials

were carried out on eppendorf tubes. Equal quantity of precursor (0.5 ml) was taken

in five different tubes and varying quantities of reducing agent (10 ll, 20 ll, 30 ll,40 ll, 50 ll) was added. 50:1 (precursor to reducing agent ratio) was identified as

the optimum ratio for proper biosynthesis of the silver nanoparticles.

Microfluidics based Handheld Nanoparticle Synthesizer 1203

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Microfluidic Device Fabrication

The microfluidic devices used in the experiments were fabricated using standard

photo-lithography followed by soft-lithography processes. The master mould was

prepared by patterning SU8 2100 and SU-8 2150 on a p-type silicon substrate to

obtain channel heights of 100 and 230 lm respectively. Microfluidic chip with

100 lm channel height was employed for carrying out the flow rate optimization

studies. A channel depth of 230 lm was used for the final negative suction based

synthesizer. The fabrication parameters were taken from the SU-8 datasheets [18].

The master moulds were then subjected to surface profiling to ascertain the channel

height. The fabricated master had a height of 133 lm while the final device had a

depth of 240 lm. Replica moulding was carried out by pouring the mixed Sylgard

184 and curing agent (10:1 ratio) on the fabricated master and curing for 2 h at

75 �C. After curing, the PDMS was peeled-off, the devices were cut and bonded to

cleaned cover glass using a plasma cleaner system.

Microfluidic Synthesis of Silver Nanoparticles

Microfluidic synthesis requires the controlled mixing of appropriate quantities of the

reactants. This is achieved by using a planar micro-mixer where the precursor and

plant extract are feed to the respective inlets as shown in Fig. 1. The expansion

chambers and the teeth pattern on the micro-mixer channel walls exploit advective

effects to cause alterations in the flow streams. This expedites the mixing process

due to the generation of multiple vortices at various spatial locations of the micro-

Fig. 1 Microfluidic micro-mixer design used for the on-chip biosynthesis of silver nanoparticles


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mixer. For finding the optimal volumetric flow rates required for the microfluidic

synthesis of nanoparticles, the precursor (silver nitrate) solution (1 mM) and pH

adjusted leaf extract are pumped through the corresponding inlet channels using

syringe pumps (NE300 New Era syringe pumps) fitted with 1 mL syringes (Fig. 2a).

Keeping the ratio between the precursor and the leaf extract as 50:1, experiments

were repeated at different flow rates (sheath flowrates of 50, 250, 500, 1000 and

2000 ll/min). This study was carried out to find the effect of different flow rates on

the synthesized nanoparticles.The Reynolds number was computed using the

following formula, R = qVD/l, where q and l are density (1000 kg/m3) and

dynamic viscosity (8.9 9 10-4 PaS) of water respectively. V is the flow rate in m/s

and D (200 um) is the hydraulic diameter defined as the ratio of four times the cross

sectional area of the pipe to the wetted perimeter of the pipe. The value of V varies

with the volumetric flow rate (50–2000 m3/min) and hence a range of Reynolds

numbers were found (3.5–150) which is well within the laminar flow.

Handheld Nanoparticle Synthesizer

The nanoparticle synthesizer is realized by employing suction based microfluidics

and appropriate modeling of the microfluidic device geometry. As an alternative to

using bulky syringe pumps, here fluid flow is achieved by the negative differential

pressure created using a single syringe (Merit Vac lock syringes) connected to the

outlet of the mixer. The inlet tubings are kept well immersed inside eppendorf tubes

which are prefilled with the reactant fluids. The eppendorfs (inlets) are exposed to

atmospheric pressure. The fluid flow is initiated by pulling the piston of the syringe

connected to the outlet and locking it to create a vacuum (low pressure). This

differential pressure induces volumetric flow rate in proportionate to the designed

hydraulic resistance of the device (Fig. 2b).

Characterization of the synthesized nanoparticles by UV–VIS absorption spectra,

X-ray Photoelectron Spectroscopy, High Resolution Transmission Electron

Microscopy and Fourier Transformation Infrared spectroscopy.

The optimization of the precursor to leaf extract ratio, and flow rates are done by

way of analyzing the Surface Plasmon Resonance (SPR) band of the synthesized

Fig. 2 Experimental setups using a syringe pumps and b negative pressure based pumping for theon-chip biosynthesis of nanoparticles


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silver nanoparticles. The microliter volumes of silver nanoparticles were taken in a

96 well plate and subjected to UV–VIS absorption spectroscopy scan from 300 to

800 nm using the TECAN 200 INFINITE plate reader.

Size and shape of the synthesized nanoparticles were confirmed by using High

Resolution Transmission Electron Microscopy (HRTEM) of FEI F20 TECNAI

TEM at size scales of 200, 100, 20 and 5 nm. Samples were prepared for HRTEM

by adding 50 ll of the nanoparticle sample on carbon coated copper grid (400 mesh)

and dried under vacuum for an hour. Fringe pattern and Energy Dispersive X-ray

Spectroscopy (EDS) analysis was obtained by FEI F30 TECNAI TEM exhibiting

the elemental composition of the nanoparticles with weight and atomic percentage.

X-ray Photoelectron Spectroscopy (XPS) was obtained by coating on-chip

synthesized silver nanoparticles on a silicon surface and examined for the presence

of silver using an AXIS ULTRA XPS instrument. Fourier Transformation Infrared

spectroscopy (FTIR) has been analysed using Perkin Elmer FTIR from 4000 to

400 nm. FTIR analysis helps to identify the interaction of nanoparticles and to

identify the possible biomolecules responsible for the capping and efficient

stabilization of the nanoparticles synthesized using leaf extracts. Prepared leaf

extract and synthesized nanoparticles solution were used as such to prepare samples

for FTIR analysis by KBr pellet technique.

Results and Discussion

The biosynthesis protocol optimizations suitable for microfluidic in situ synthesis

were carried out using UV–VIS absorption studies. Initial off chip synthesis was

carried out to estimate the desired ratio of silver nitrate and leaf extract for the

formation of nanoparticles. In comparison to different ratios, 50:1 (silver nitrate:

leaf extract) yielded a hump in the absorption studies (Fig. S1 in supplemental

material). However, the obtained Surface plasmon resonance (SPR) peak was

broader and the absorption value was lower indicating low yield of nanoparticle.

This was overcome by maintaining the pH of the reaction at 10 by the addition of

NaOH to the reducing agent.

The SPR peak for the off-chip synthesis was at 420 nm. Keeping the ratio fixed

to 50:1, on-chip synthesis studies were subsequently performed. Formation of silver

nanoparticles can be visually observed by the color change starting from the

junction of the two inlets to the outlet of the microfluidic chip as seen in Fig. 3a, b

of pumping. In the same ratio, different volumetric flow rates (50:1 lL/min,

250:5 lL/min, 500:10 lL/min, 1000:20 lL/min, 2000:40 lL/min) were tried and

the SPR peak has been found to be varying, which can be attributed to change in

size of nanoparticles getting synthesized (Fig. 4). At lower flow rates the peak is

found to be around 432 nm while at higher flow rates of 1000:20 lL/min,

2000:40 lL/min, the SPR peak lies at 418 nm. This has also led to the synthesis of

monodisperse and larger number of nanoparticles as evident through the sharper and

larger amplitude SPR peaks (Fig. 4). The SPR peak is blue shifted with higher flow

rates which are attributed to the smaller size of particles being present in the


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nanoparticles generated with higher flow rates. Thus the size of the nanoparticles

can be tuned by appropriate changes in the volumetric flow rates of the reactants.

Once the presence of silver nanoparticles was confirmed from the UV–VIS

studies, HRTEM was carried out to examine the morphology of the on-chip

(microfluidic) synthesized nanoparticles. Spherical and evenly distributed nanopar-

ticles were found in HRTEM images taken at different scales (Fig. 5a–d). Fringe

pattern observed at 5 nm scale is analyzed using Image J and the pitch of the pattern

is found to be 0.23 nm (Fig. 5e), which matches with the d-spacing of silver

nanoparticles for the crystal lattice plane [111]. Selected area electron diffraction

(SAED) pattern was obtained for the nanoparticles showing distinct rings having a

matching d-spacing value of 0.235 nm for crystal lattice plane [111] and 0.144 nm

for crystal lattice plane [220] (Fig. 5f).

Fig. 3 Optical Microscope image of the reddish brown solution of the nanoparticles formed at thejunction of inlet micro-channels carrying silver nitrate and Parthenium histerophorus leaf extract in themicrofluidic reactor (Color figure online)

Fig. 4 UV–VIS Spectroscopy graph exhibiting a shift in wavelength of the SPR peak of silvernanoparticles synthesized using microfluidic reactor as the flow rate is varied


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By analyzing TEM image of the silver nanoparticles using MATLAB, it is found

that the majority of the nanoparticles size lies between 4 and 6 nm as is shown in the

histogram (Fig. S2). EDS analysis shown in Fig. 6 and adjacent table, indicate the

presence of silver with very less oxygen (about 0.38% by weight). This negligible

presence indicates the formation of metal nanoparticles and minimal contamination

Fig. 5 Different scales of Transmission Electron Microscopy images of silver nanoparticles synthesizedusing the microfluidic reactor at scales of a 200 nm, b 100 nm, c 20 nm, d 20 nm, e 5 nm, f 2.5 andSAED pattern


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due to metal oxide. Also, the low presence of nitrate is due to the unpurified

nanoparticles which were as such used for EDS analysis. Additionally comparision

of Transmission Electron Microscopy images of on-chip synthesized silver

nanoparticles at different flow rates of silver nitrate to leaf extract at 500:10,

500:50 and 500:100 ll/min were also carried out and the images were depicted in

Fig.S3. XPS analysis of on-chip synthesized silver nanoparticles has been carried

out and the peaks for silver 3d3/2 and 3d5/2 have been obtained (Fig. 6b) which

confirms presence of silver.

Mechanism of On-Chip Microfluidic Biosynthesis

Silver nanoparticle synthesis has been carried out through a continuous flow process

on a microfluidic chip. Parthenium histeroporus contain Caffeic acid (phenolic

acid) and Parthenin as a major phytochemical constituent as reported by Das et al.

1995 [19]. These phytochemicals are responsible for the formation of silver

Fig. 6 a Energy Dispersive X-ray spectroscopy (EDS) graph of silver nanoparticles synthesized usingmicrofluidic reactor and insettable showing the percentage composition of different elements, b X-rayPhotoelectron Spectroscopy graph of silver nanoparticles exhibiting peaks for Ag 3d3/2 and 3d5/2

Fig. 7 Reducing mechanism of the biosynthesis of silver nanoparticles using microfluidic reactor


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nanoparticles as described in the schematic representation (Fig. 7). The reducing

mechanism of biosynthesis of metal nanoparticles have been analysed by means of

Fourier Transformation Infrared spectroscopy (FTIR) of the leaf extract, and

synthesized silver nanoparticles (Fig. S4). Table S1 displays the bands observed in

the leaf extract and the metal nanoparticles and the possible functional groups

involved in the formation of the metal nanoparticles. The bands observed at

1636 cm-1 in the leaf extract arises from carbonyl group and it is shifted to

1637 cm-1 in the nanoparticles suggesting that carbonyl groups present in the leaf

extract might have interacted with the nanoparticles. The band at 3400–3500 cm-1

is characteristic of the O–H stretching vibration of the alcoholic compounds.

The observation of bands at 1405 and 1328 cm-1 in the leaf extract and the slight

shift of these bands in the nanoparticles may be attributed due to the C–O, C–N

stretching vibrations of the alcohols and the aromatic amine groups. The bonds or

functional groups such as –C–C–, –C–O–, and –C–O–C– are derived from the

compounds present in the leaf extract of Parthenium histerophorus. It may be

assumed that water soluble compounds such as flavanoids, terpenoids are the

capping ligands of the nanoparticles. The band at 1257 cm-1 confirms the presence

of C–O groups from polyols which may have played crucial role in the synthesis of

nanoparticles. The shift of this band can be attributed to the reduction of metal ions

coupled with the oxidation of phenolic components of polyols. The band at

672 cm-1 in the leaf extract was shifted to 670 cm-1 and the decrease in the

transmittance in the nanoparticles indicates the possible involvement of some

aromatic compounds along with the polyols present in the leaf extract in the

reduction of metal ions.

Handheld Nanoparticle Synthesizer

The optimized ratio and flowrates obtained earlier were used to develop the

handheld synthesizer. The synthesizer module encompasses a custom micro-mixer

and a pumping mechanism which have been designed to suit on-site synthesis. The

micromixer design with desired channel dimensions and the electrical analogy

model are shown in Fig. 8. The initial microfluidic design used for conducting the

optimization studies has identical channel lengths and width for feeding the two

Fig. 8 Design for the handheld on-chip biosynthesis of silver nanoparticles using suction microfluidics


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reactants. The different ratios of the precursor and the reducing agent was obtained

by flowing them using two syringe pumps set at different desired flow rates. The use

of bulky syringe pumps can be eliminated by flowing the reactants using a

differential pressure created using a single 20 ml negative pressure syringe.

By using the electrical circuit-pressure driven microfluidics analogy [20], the

dimensions aremodified based on the hydraulic resistance calculations and the applied

differential pressure. The hydraulic resistance is computed individually for each of the

two inlet channels and the micro-mixer section of the microfluidic device. Given the

channel width w, height h, length of the channel section L and the viscosity of the fluid

flowing g, the hydraulic resistance is found using the Hagen–Poiseuille’s law [21].

R ¼ 12gLwh3

ð1Þ

As the hydraulic resistance is inversely proportional to the volumetric flowrate,

the resistance ratio of the precursor inlet channel to the leaf extract inlet channel

should be 1:50 to obtain a 50:1 dilution ratio. Using Eq. (1), the resistance

calculated of the precursor inlet channel section, leaf extract inlet channel and mixer

channel for a channel height of 230 lm was 3.51, 175.6 and 511.3 GPas/m3

respectively. The flow rates measured with the device were around 1200 lL/min.

The SPR of the synthesized nanoparticles using the off-chip and the onchip negative

suction based methods exhibited peaks at 420 nm (Fig. S5). Although both the

Fig. 9 Comparision of High Resolution Transmission Electron Microscopy images off-chip and on-chipsynthesized silver nanoparticles at different scales (100 and 50 nm)


123

methods have a peak at the same wavelength, there is a narrower SPR absorbance

peak for silver nanoparticles synthesized using the on-chip method than off-chip

synthesized nanoparticles. This attributes to the better formation of the silver

nanoparticles by negative suction based method of synthesis.

A comparison of HRTEM images of nanoparticles synthesized off-chip, on-chip

with syringe pumps and on-chip suction based are compared in Fig. 9. HRTEM

studies show that on-chip suction based biosynthesis produces more monodisperse

silver nanoparticles while off-chip biosynthesis generates Ag particles of varying

sizes. The Nanoparticle Synthesizer assembly with a 3D printed casing using a

Wanhao duplicator 4 is seen in Fig. 10.

Conclusions

Present study has led to the realization of two main objectives: (i) optimized

protocol for room-temperature negative suction microfluidics based biosynthesis of

monodisperse silver nanoparticles and (ii) a handheld microfluidic nanoparticle

synthesizer suitable for field applications. Current nanoparticle biosynthesis

protocol was developed by optimizing the ratio of precursor to reducing agent as

50:1 by analyzing SPR absorption peaks. A microfluidic device capable of mixing

desired volumes of precursor and reducing agent at suitable flowrates has been

designed. The fluid pumping is accomplished by exploiting the differential pressure

created using a syringe connected to the outlet of the device. Hence, the microfluidic

synthesis reported here has made possible the realization of a robust and compact

synthesizer of nanoparticles by providing a great control on the micro-volumes of

the reagents to be mixed at an optimal time-scales. The microfluidic negative

Fig. 10 Design and 3D printed prototype of the nanoparticle synthesizer


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suction synthesized nanoparticles exhibited narrow size distribution of 10 nm

compared to the previous report of polydispersed off-chip synthesized nanoparticles

using the same Parthenium leaf extract [17]. Thus a handheld electrical power free

microfluidic nanosynthesizer has been designed and developed for room temper-

ature synthesis of nanoparticles. The nanoparticles synthesized using the synthesizer

can be used for variety of applications such as sensing at secluded environments.

Supplementary Material

See supplementary material for nanoparticle characterization data (UV–VIS

Absorbance spectroscopy optimization graph, TEM images for different flow rates,

FTIR graph, Particle Size Histogram, and a table containing different FTIR bands).

Acknowledgements The authors gratefully acknowledge SPARSH: Social Innovation Programme for

Products: Affordable and Relevant to Societal Health project funded by BIRAC-Biotechnology Industry

Research Assistance Council for the financial assistance. Authors acknowledge Robert Bosch Centre for

Cyber Physical System for their support. Also, the authors thank Mr.JayeshAdhikari for his assistance in

designing and 3D printing the nanoparticle synthesizer prototype casing.

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microfluidics based handheld nanoparticle synthesizer · original paper microﬂuidics based...

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