- Email: [email protected]
Piezoresistive microcantilever based lab-on-a-chip system for detection of macronutrients in the soil
Accepted Manuscript Piezoresistive microcantilever based lab-on-a-chip system for detection of macronutrients in the soil Rajul S Patkar, Mamta Ashwin, V Ramgopal Rao PII: DOI: Reference:
S0038-1101(17)30534-8 http://dx.doi.org/10.1016/j.sse.2017.07.007 SSE 7278
To appear in:
Solid-State Electronics
Please cite this article as: Patkar, R.S., Ashwin, M., Ramgopal Rao, V., Piezoresistive microcantilever based labon-a-chip system for detection of macronutrients in the soil, Solid-State Electronics (2017), doi: http://dx.doi.org/ 10.1016/j.sse.2017.07.007
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.
Piezoresistive Microcantilever based Lab-on-aChip System for Detection of Macronutrients in the Soil Rajul S Patkar, Mamta Ashwin, V Ramgopal Rao Department of Electrical Engineering Indian Institute of Technology Bombay, Mumbai, India Email: [email protected]; [email protected] Abstract— Monitoring of soil nutrients is very important in precision agriculture. In this paper, we have demonstrated a micro electro mechanical system based lab-on-a-chip system for detection of various soil macronutrients which are available in ionic form K+, NO3-, and H2PO4-. These sensors are highly sensitive piezoresistive silicon microcantilevers coated with a polymer matrix containing methyltridodecylammonium nitrate ionophore/ nitrate ionophore VI for nitrate sensing, 18-crown-6 ether for potassium sensing and Tributyltin chloride for phosphate detection. A complete lab-on-a-chip system integrating a highly sensitive current excited Wheatstone’s bridge based portable electronic setup along with arrays of microcantilever devices mounted on a printed circuit board with a liquid flow cell for on the site experimentation for soil test has been demonstrated. Keywords—MEMS, Agricultural sensors, microcantilever
I.
I N T R O D U C TIO N
To increase agricultural production, excessive chemical fertilizers are added to the soil. This unnecessary addition of fertilizers contaminates surface and ground water which causes an undesirable environmental impact and an unnecessary increase in the cost of production. Systems are available based on different principles like Ion Selective Electrode (ISE), Ion Selective Field Effect Transistor (ISFET) and other spectroscopic principles based on reflectance measurement in the visible, near and mid-infrared ranges for detection of nitrates, phosphates and potassium (NPK) in the soil. A few spectroscopy based methods which depend on colorimetric measurements are ex-situ and need soil sample preparation, whereas other spectroscopic based sensors are in-situ but are very expensive. The conventional way of testing soil nutrients is to extract nutrients from the soil by shaking it in standard reagent solution and then filtering the suspension to get the soil solution. This solution is then measured either with Inductively Coupled Plasma – Atomic Emission Spectroscopy (ICP-AES) or Atomic Absorption Spectroscopy (AAS) [1]-[5]. There is still not a single lab-on-a-chip (LOC) system available commercially in the market for soil nutrients. Hence, a low cost handy tool for nutrient sensing which is reliable and simple would be useful to design smart irrigation and fertilization management systems. This paper demonstrates our work in the direction of developing a complete LOC system for soil nutrients based on a highly sensitive piezoresistive cantilever platform. The schematic representation of a piezoresistive microcantilever based LOC system is shown in fig. 1(a). A reference and three different test microcantilevers functionalized with the coatings of NPK will selectively bind different nutrients and will result in a change of resistance as shown in the fig. 1(b & c). This change in resistance will be measured by highly sensitive electronics. The microcantilever based sensor systems have the advantages of being robust, small, versatile and scalable which enable large-scale deployment. The sensors also provide a quick response, consume less power and have low fabrication costs because of batch processing [6]-[8].
Fig.1 Schematic representation of (a) a lab-on-a-chip system (LOC) (b) sensing principle of a micro cantilever (c) microcantilever array depicting the sensing of
II. F AB R IC A T IO N O F M IC R O C AN T ILEV E R PL ATF O R M A microcantilever platform has been used in various areas of biosensing like point of care (POC) diagnostics, drug discovery [9], explosive detection [10], [11], environmental sensing and mass spectrometry but it has not been used in agricultural applications for detection of macronutrients. The advantage is that microcantilevers can be used in various diverse ways depending on applications such as vacuum, liquid or gas phase. The sensing principle can be either static mode where the deflection of the device is measured or dynamic mode where change in the resonant frequency signifies the sensing. Different materials and methods are used for fabricating microcantilevers depending on the applications. Silicon and its compounds, polymers and metals are generally used to fabricate these devices. The advantage of a polymer microcantilever is its high sensitivity because of lower Young’s modulus but these materials have stability problems in liquid [8]. Silicon nitride is the most reliable material for sensing in agricultural applications as it involves ionic solutions [12].
Fig. 2 (a)-(f) Process flow for highly sensitive piezoresistive silicon microcantilever fabrication with a total stack thickness of 900 nm (a) Double sided polished wafer with SiO2 on both sides (b) Structural layer nitride deposited and patterened (c) Poly-silicon layer deposition (doped poly) and patterning (d) Encapsulation layer silicon nitride deposition and patterning (opening of contact window) for deposition of contact pads (e) patterning of contact pads (f) silicon etching using DRIE to form cavity (g) Optical Image of a released microcantilever die (h) SEM image of a released microcantilever
Even though silicon based materials are suitable for sensing in liquid medium, the high Young’s modulus make them less sensitive. The geometry of these devices should be planned to achieve high sensitivity and gauge factor. The thickness of the microcantilever should be small for achieving higher surface stress sensitivity. The individual thickness of each layer of the stack should be controlled to have the piezoresistive layer away from the neutral axis so that maximum surface stress can be transferred to the piezo layer. The high strain leads to large change in delta R resulting in higher gauge factors. The microcantilevers used in this work were fabricated using bulk micromachining. The microcantilevers have an overall stack (Silicon nitride-doped polysilicon-Silicon nitride) thickness of 900 nm, a length of 250 µm and width of 110 µm while having a spring constant of 0.2 N/m. The fabrication process started with an RCA cleaned double side polished (DSP) silicon substrate. SiO2 was deposited on both sides of the substrate. Bottom layer of SiO2 acted as a mask for etching silicon and top layer of SiO2, as an etch stop layer while etching silicon. A stack of silicon nitride, doped polysilicon and silicon nitride were deposited and patterned one by one. The bottom structural layer was made of 700 nm thick silicon nitride, deposited by Low Pressure Chemical Vapour Deposition (LPCVD) process and patterned. A piezoresistive layer of boron doped polysilicon (100 nm) was deposited and patterned. The top encapsulation layer is also of silicon nitride of thickness 100 nm. Windows were opened in the top silicon nitride, to create contacts to piezoresistor. Metal was deposited and patterned. The back side silicon oxide was patterned and silicon was etched using deep reactive ion etching (DRIE) process to form the pit so that the microcantilevers could be suspended. The top oxide was later etched using buffered hydrofluoric acid (BHF). The fabrication steps and SEM image of the released microcantilever is shown in fig. 2. We have also designed and fabricated a cost effective piezoresistive silicon nitride microcantilever platform with polymer anchor based on SPARED (Spin, Pattern, Anchor and Release Devices) MEMS process [13]. This process combines the advantage of surface micromachining of polymer with the material stability of silicon nitride (fig. 3). The device was characterized for all electrical, mechanical and electro-mechanical parameters. The microcantilever structure has a total stack thickness of 1075 nm with a spring constant of 0.9 N/m, deflection sensitivity of 0.3 ppm nm-1 and a gauge factor of 22. The resonant frequency and quality factor of this device is 22.5 KHz and 28 respectively as measured in air [14].
Fig. 3 (1) Fabrication sequence of piezoresistive nitride microcantilever with polymer anchor (a) substrate with sacrificial layer (b) structural layer silicon nitride of thickness 650 nm (c) patterned structural layer (d) patterned p-polysilicon on structural layer (e) patterned encapsulation nitride layer with contact window (f) patterned titanium /gold contact pads (g) polymer anchor (h) released microcantilever die (i) Optical image of a released die of silicon nitride microcantilever with polymer anchor (j) optical image of a released microcantilever die (2) (a) Plot of force-deflection curve from nanoindentation (b) Resonant frequency plot (c) Plot of ΔR/R as a function of deflection
III. P R IN C IP LE O F S E N SIN G AN D M ETH O D O LO G Y A microcantilever based platform having array of sensors is capable of determining several analytes, such as soil macronutrients and pH simultaneously. Polymeric ionophore doped ion-selective membranes were created using Polyvinylchloride-Dioctylsebacate (PVC-DOS) matrix which acts as a hydrophobic phase. Plasticizer like DOS is used to help in easy diffusion of ionophore in chelating the ions. It has been reported that this PVC-DOS mixture creates a homogeneous hydrophobic medium to facilitate the diffusion of ionophore, ion complexes similar to water-immiscible organic solvent. This polymeric matrix also improves mechanical stability [2]. To coat the microcantilevers with the ionophores, it is required that the ionophores are embedded in a polymer matrix. The analyte salt solution will remove the coatings of ionophore in absence of polymer matrix. In all our experiments, the membranes were prepared with the mixture consisting of 4 wt% ionophore, 66 wt% DOS and 33 wt% PVC. Since the microcantilever surface needs to be functionalized with this mixture, the thickness of the film is optimized by diluting it with tetrahydrofuran (THF). The porosity and roughness of the film were characterized by Atomic Force Microscope (AFM). The AFM image of the optimized film is shown in fig. 4-1. The cross-section Scanning Electron Microscope (SEM) image of the film shows the thickness to be 25nm as shown in fig. 4-1(d). Ionophores in the polymer matrix of PVC and DOS were asymmetrically immobilized on the microcantilever surface for lab-on-chip-detection of soil macronutrients NPK. Sensor array can also be helpful in improving the selectivity by having multiple sensors coated with different receptor analytes and decision can be made on sensing using a pattern recognition algorithm. Given below are the specific ionophores used for NPK detection. Potassium Sensing: Ionophores like Valinomycin [4] and 18-crown-6 ether [15], [16] have been reported in the literature for potassium sensing. ISE and ISFETs have used Valinomycin based PVC and plasticizer membranes for potassium ion sensing. Here we report use of 18-crown-6 ether as potassium ionophore for our piezoresistive micocantilever based sensing experiments. The schematic representation of the coating and the host guest interaction of crown ether with the potassium ion on microcantilever surface is shown in fig. 4-2 (c & d). Nitrate Sensing: Similarly, for nitrate sensing, we have used methyltridodecylammonium nitrate (MTDAN) and nitrate ionophore VI in polymer matrix [17]. Phosphate Sensing: Sasaki et al. have shown that the ion selective membrane containing tributyltin chloride as the ionophore exhibited high selectivity for H2PO4 with the addition of 25 mol% sodium tetrakis-[3,5- bis(trifluoromethyl)phenyl]borate (NaTFPB) [18]. For lab-on-a-chip detection of phosphates, we have used the same composition of ionophore and additive in the polymer matrix.
Fig. 4 (1) 3D AFM micograph of a drop casted surface showing good porosity which is required for diffusion of ions into the film for sensing purpose (b) 2D micrograph of the film (c) plot showing the height profile versus scanned distance (d) Cross section Scanning Electron Microscope (SEM) image of the film (thickness 26 nm), bottom silicon dioxide thickness 110 nm (2) (a) Molecular representation of 18-crown-6 ether (b) Molecular representation of 18-crown-6 ether with potassium ion in the cavity (c) Schematic representation of 18-crown-6 ether immobilized on microcantilever surface in a PVC/DOS matix (d) Schematic representation of potassium ion trapped in crown ether cavity resulting in deflection of microcantilever
IV. E X PE R IM EN TA L SE T - U P FO R LAB - O N - A - C H IP S O IL N U T R IEN T AN A LY S IS A microcantilever die consists of an array of four microcantilevers as shown in fig. 5 (a). This was mounted on a printed circuit board (PCB) and contacts were made using either wire bonding or silver epoxy. A Teflon flow cell with inlet and outlet was placed on the PCB. Out of four microcantilevers, one was used as a reference and the other three were test cantilevers used for sensing. The test microcantilevers are coated with receptor molecules embedded in a polymer matrix of PVC and DOS. The reference microcantilever is coated with only PVC and DOS to nullify the effect of soil solution on PVC/DOS polymer matrix. The surface of the microcantilever is asymmetrically immobilized with the polymer matrix using either a nano-dispenser or micropipette as shown in fig. 5 (b). The asymmetric coating of microcantilever is shown in fig. 5 (e & f). Fig 5 (e) shows the top side of the microcantilever coated with a PVC/ DOS based matrix along with a specific ionophore. A complete experimental set up is shown in fig. 5 (d). Microcantilever mounted on a PCB which is connected to a four channel resistance measurement setup is shown in fig. 5 (f). Once the devices are coated with ionophore embedded polymer, cured and packaged, these cartridges can be used for few months without any problem. Once the devices are used, they can’t be reused.
Fig. 5 (a) A microcantilever die (b) Coating an individual microcantilever with the help of a nano dispenser (c) microcantilever mounted in flow cell (d) complete experimental set-up (e) microcantilever coated with a PVC/ DOS based matrix along with a specific ionophore demarcating the extent to which the microcantilever is coated (f) microcantilever when viewed on the reverse side (g) A PCB connected to a four channel measurement system
Initial focus was to sense individual analytes with different ionophores. Further, the experiments were extended to sense multiple analytes and also study the effect of interfering ions. The experiments were carried out with two different methodologies. In the first method, 100 µl of 1 mM KCl solution was introduced at a constant flow rate using a syringe pump. In the second case, 100 µl of DI water was introduced into the flow cell by using a micropipette. The introduction of fluid causes a jump in resistance. Once the resistance values are steady in fluid, 10 µl of known concentration of KCl solution was introduced in the flowcell. 10 µl of sodium nitrate solution was introduced for nitrate sensing. V. R ESU LT S AN D D IS C U SS IO N Ionophores, named earlier as ion complexes are basically charge carriers or charge bearers. These molecules have a hydrophilic interior which binds specific ions and a hydrophobic exterior that interacts with the hydrocarbon interior of the membrane. Most ionophores form a cyclic ring concentrating oxygen and nitrogen functional groups at the center of their structure to associate with the cation or anion. The size of the cavity determines which ion can be encapsulated [19]. The six oxygen atoms of the 18-crown-6 ether interact with the bound K+ ion and these interactions are purely electrostatic. The sodium ions are smaller in size and they cannot interact at a time with six oxygen atoms and thus are not energetically favorable. The concentration gradient between organic film and aqueous phase helps the diffusion of the analyte ions through the film. The complexation of an ion in an ionophore results in conformal changes which in turn generates the stress on the microcantilever surface. Also since ionophores are charge bearers, the complexation of ion in ionophore results in charge separation layer of few nanometers. Since the microcantilevers are asymmetrically coated, it leads to accumulation of charges on one side and the conformal changes of ionophore results in the deflection of the microcantilever [20], [21]. The schematic of the same is depicted in fig. 6 (a). The deflection of the microcantilever generates the strain in the piezoresistive layer of the microcantilever resulting in change of resistance. Changes in resistances of all the microcantilevers were recorded using a four channel measurement system. The block diagram of the schematic of the measurement setup is shown in fig. 6 (b).
Fig. 6 (a) Schematic diagram depicting the charge accumulation and double layer formation on one side of the surface resulting in deflection (b) Block diagram of the portable system for point of use agriculture application
The plots of fig. 7 (a), 7 (b) and 7 (c) show the results of potassium sensing using 18-crown-6 ether. In fig. 7 (a), 1 mM of KCl was introduced into the flow cell at 6th min and the change in resistance was plotted with respect to time. In case of fig. 7 (b) and (c), after the resistance achieves a steady value in DI water, a known amount and known concentration of KCl is added resulting in 1 pM effective concentration of KCl solution in first case and 45 µM in second case.
Fig. 7 Sensor response to asymmetrically coated microcantilever with 18-crown-6 ether to potassium ions
Two different ionophores, nitrate ionophore VI and MTDAN were used for nitrate sensing. The fig. 8 (a & b) shows the response to nitrate ion analytes. In 8 (a), nitrate solution was introduced after the microcantilevers were stable in DI water and for 8 (b), the nitrate solution was introduced in the beginning itself. The microcantilever which was coated with nitrate ionophore showed the response to the analyte salt solution whereas the reference microcantilever which was just coated with polymer matrix did not show any response.
Fig. 8 Plots of nitrate sensing (a) Sensor response of microcantilever asymmetrically coated with nitrate ionophore VI in polymer matrix to nitrate ions (b) Sensor response of microcantilever asymmetrically coated with MTDAN in polymer matrix to nitrate ions.
Control experiments were carried out to confirm the selectivity and sensitivity of the ionophores. In the first set of experiments, the test microcantilever was functionalized with 18-crown-6 ether in a polymer matrix as described previously. Once the microcantilevers were stable in DI water, a known amount and concentration of sodium nitrate was introduced in the flowcell in the 13th minute. As seen in fig. 9 (a) the microcantilevers coated with 18-crown-6 ether did not show any response to the nitrate ions. In the second experiment, the test microcantilever was functionalized with MTDAN in a polymer matrix. A known amount and concentration of KCl was introduced in the flowcell in the 12th min after stabilization of the microcantilevers. As seen in fig. 9 (b) the microcantilevers coated with MTDAN did not show any response to the potassium ions. This shows that the ionophores are highly selective to particular ions.
Fig. 9 Plots of ionophore subjected to interfering ions (a) Sensor response of microcantilever asymmetrically coated with 18-crown-6 ether (potassium ionophore) in polymer matrix to nitrate ions (b) Sensor response of microcantilever asymmetrically coated with MTDAN (nitrate ionophore) in polymer matrix to potassium ions.
V I. C O N C LU S IO N As part of this work, we have demonstrated for the first time the measurement of soil macronutrients (NPK) using MEMS based lab-on-a-chip system. Sensing of potassium and nitrates has been demonstrated using 18-crown-6 ether, MTDAN and nitrate ionophore VI embedded in a polymer matrix. Similarly, we could successfully create and asymmetrically coat phosphate selective ionophore polymeric matrix on to the microcantilever. Currently, we are working on the phosphate sensing experiments. This platform can be further extended to test various other soil parameters like soil micronutrients and pathogens. A low-cost silicon nitride microcantilever platform is also developed for the agricultural applications. VII. A C K N O WLED G M EN T This work was supported by the Department of Science and Technology, Department of Electronics and Information Technology, Government of India, through the Centre of Excellence in Nanoelectronics. We would like to thank Dr. Madhuri Vinchurkar for the discussions.
R E FE R EN C ES [1] [2] [3] [4]
H.J. Kim et al., “Soil macronutrient sensing for precision agriculture,” J. Environ. Monit, vol. 11, pp. 1810–1824, 2009 Philippe Buhlmann and Li D. Chen, “Ion-Selective Electrodes With Ionophore- Doped Sensing Membranes”, In Supramolecular Chemistry: From Molecules to Nanomaterials, John Wiley & Sons, , 2012 Akshay Sankpal and Krishna K. Warhade, “Review of Optoelectronic Detection Methods for the Analysis of Soil Nutrients”, International Journal of Advanced Computing and Electronics Technology, Vol. 2, No. 2, 2394-3416, 2015 H.J Kim et al, “ Evaluation of nitrate and potassium ion-selective membranes for soil macronutrient sensing”, Transactions of the ASABE, Vol. 49(3), pp 597-606, 2006
[5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18] [19] [20] [21]
J. R. Brown and R.R Rodriguez, “ Soil Testing in Missouri- A Guide for conducting soil tests in Missouri”, University of Missouri, Extension Circular No 923, 1983 Nickolay V. Lavriket et al., “Cantilever transducers as a platform for chemical and biological sensors”, Review of scientific instruments, Vol. 75, No. 7, pp. 2229-2253, 2004 R. S. Patkar, M. G. Seelan, and V. R. Rao, “A highly sensitive piezoresistive cantilever based sensor platform for detection of macronutrients in soil,” in Proc. IEEE- NANO, pp 751-754, Jul 2015. Anja Boisen et al, “ Cantilever like Micromechanical Sensors”, Rep. Prog. Phys, Vol. 74, pp30, 2011. Sen Xu and Raj Mutharasan, “ Cantilever Biosensors in drug discovery”, Informa Healthcare, Vol.4, No 12, pp1237-1251, 2009. Larry Sensac and Thomas Thundat, “Nanosensors for explosive detection”, Materials Today, Vol.11, No. 3, pp 28-36, March 2008. V. Seena et al, "Polymer Nanocomposite nanomechanical cantilever sensors: Material characterrization, device development and application in explosive vapour detection”, Nanotechnology, Vol.22, No. 29, pp 11, 2011. S. M. Ali et al, “Mechanical Performance of cantilevers in liquids”, J. Microelectromech. Syst., Vol 20, No.2, pp 441-45, April 2011. Rajul S. Patkar, et.al., “Polymer-Based Micro/Nano Cantilever Electro-Mechanical Sensor Systems for Bio/Chemical Sensing Applications” Micro and Smart Devices and Systems, Editors: K.J. Vinoy, G.K. Ananthsuresh, Rudra Pratap, S.B. Krupanidhi, Springer Tracts in Mechanical Engineering 2014, pp 403-422, ISBN 978-81-322-1912-5(Print) 978-81-322-1913-2(online) Rajul S Patkar et al., “ A novel SU8 polymer anchored low temperature HWCVD nitride polysilicon piezoresistive cantilever”, IEEE J-MEMS, Vol. 23, No.6, December 2014 Mohammed Reza Ganjali et al, “Supramolecular Based Membrane Sensors”, Sensors, Vol. 6, No.8, pp1018-1086, 2006. Chang Min Choi et al., “Binding selectivity of dibenzo-18-crown-6 for alkali metal cations in aqueous solution: A density functional theoy study using a continuum solvation model”, Chem. Cent. J, vol. 6, No. 84, pp. 8, 2012. J. Gallardo et al, “A flow-injection electronic tongue based on potentiometric sensors for the determination of nitrate in the presence of chloride”, Sensors and Actuators B: Chemical, Vol. 101, No.1, pp 72-80, 2004 S. Sasaki et al, “ Organic Tin Compounds combined with anionic additives- an ionophore system leading to a phosphate ion selective electrode” Talanta, Vol. 63, pp 131-134, 2004 Manihiro Hillary, “Mechanism of action of ionophores, examples and their significance in a cell”, Slideshare (http://www.slideshare.net/ManirihoHillary/ionophores), 2016 Neng-Hui Zhang et al, “Effect of Surface Charge State on the surface stress of a microcantilever”, Nanotechnology, 27, pp. 1-10, 2016 Thomas Thundat, United States Patent, Patent No: 6,016,686, Micromechanical Potentiometric Sensors, 2000
S0038-1101(17)30534-8 http://dx.doi.org/10.1016/j.sse.2017.07.007 SSE 7278
To appear in:
Solid-State Electronics
Please cite this article as: Patkar, R.S., Ashwin, M., Ramgopal Rao, V., Piezoresistive microcantilever based labon-a-chip system for detection of macronutrients in the soil, Solid-State Electronics (2017), doi: http://dx.doi.org/ 10.1016/j.sse.2017.07.007
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.
Piezoresistive Microcantilever based Lab-on-aChip System for Detection of Macronutrients in the Soil Rajul S Patkar, Mamta Ashwin, V Ramgopal Rao Department of Electrical Engineering Indian Institute of Technology Bombay, Mumbai, India Email: [email protected]; [email protected] Abstract— Monitoring of soil nutrients is very important in precision agriculture. In this paper, we have demonstrated a micro electro mechanical system based lab-on-a-chip system for detection of various soil macronutrients which are available in ionic form K+, NO3-, and H2PO4-. These sensors are highly sensitive piezoresistive silicon microcantilevers coated with a polymer matrix containing methyltridodecylammonium nitrate ionophore/ nitrate ionophore VI for nitrate sensing, 18-crown-6 ether for potassium sensing and Tributyltin chloride for phosphate detection. A complete lab-on-a-chip system integrating a highly sensitive current excited Wheatstone’s bridge based portable electronic setup along with arrays of microcantilever devices mounted on a printed circuit board with a liquid flow cell for on the site experimentation for soil test has been demonstrated. Keywords—MEMS, Agricultural sensors, microcantilever
I.
I N T R O D U C TIO N
To increase agricultural production, excessive chemical fertilizers are added to the soil. This unnecessary addition of fertilizers contaminates surface and ground water which causes an undesirable environmental impact and an unnecessary increase in the cost of production. Systems are available based on different principles like Ion Selective Electrode (ISE), Ion Selective Field Effect Transistor (ISFET) and other spectroscopic principles based on reflectance measurement in the visible, near and mid-infrared ranges for detection of nitrates, phosphates and potassium (NPK) in the soil. A few spectroscopy based methods which depend on colorimetric measurements are ex-situ and need soil sample preparation, whereas other spectroscopic based sensors are in-situ but are very expensive. The conventional way of testing soil nutrients is to extract nutrients from the soil by shaking it in standard reagent solution and then filtering the suspension to get the soil solution. This solution is then measured either with Inductively Coupled Plasma – Atomic Emission Spectroscopy (ICP-AES) or Atomic Absorption Spectroscopy (AAS) [1]-[5]. There is still not a single lab-on-a-chip (LOC) system available commercially in the market for soil nutrients. Hence, a low cost handy tool for nutrient sensing which is reliable and simple would be useful to design smart irrigation and fertilization management systems. This paper demonstrates our work in the direction of developing a complete LOC system for soil nutrients based on a highly sensitive piezoresistive cantilever platform. The schematic representation of a piezoresistive microcantilever based LOC system is shown in fig. 1(a). A reference and three different test microcantilevers functionalized with the coatings of NPK will selectively bind different nutrients and will result in a change of resistance as shown in the fig. 1(b & c). This change in resistance will be measured by highly sensitive electronics. The microcantilever based sensor systems have the advantages of being robust, small, versatile and scalable which enable large-scale deployment. The sensors also provide a quick response, consume less power and have low fabrication costs because of batch processing [6]-[8].
Fig.1 Schematic representation of (a) a lab-on-a-chip system (LOC) (b) sensing principle of a micro cantilever (c) microcantilever array depicting the sensing of
II. F AB R IC A T IO N O F M IC R O C AN T ILEV E R PL ATF O R M A microcantilever platform has been used in various areas of biosensing like point of care (POC) diagnostics, drug discovery [9], explosive detection [10], [11], environmental sensing and mass spectrometry but it has not been used in agricultural applications for detection of macronutrients. The advantage is that microcantilevers can be used in various diverse ways depending on applications such as vacuum, liquid or gas phase. The sensing principle can be either static mode where the deflection of the device is measured or dynamic mode where change in the resonant frequency signifies the sensing. Different materials and methods are used for fabricating microcantilevers depending on the applications. Silicon and its compounds, polymers and metals are generally used to fabricate these devices. The advantage of a polymer microcantilever is its high sensitivity because of lower Young’s modulus but these materials have stability problems in liquid [8]. Silicon nitride is the most reliable material for sensing in agricultural applications as it involves ionic solutions [12].
Fig. 2 (a)-(f) Process flow for highly sensitive piezoresistive silicon microcantilever fabrication with a total stack thickness of 900 nm (a) Double sided polished wafer with SiO2 on both sides (b) Structural layer nitride deposited and patterened (c) Poly-silicon layer deposition (doped poly) and patterning (d) Encapsulation layer silicon nitride deposition and patterning (opening of contact window) for deposition of contact pads (e) patterning of contact pads (f) silicon etching using DRIE to form cavity (g) Optical Image of a released microcantilever die (h) SEM image of a released microcantilever
Even though silicon based materials are suitable for sensing in liquid medium, the high Young’s modulus make them less sensitive. The geometry of these devices should be planned to achieve high sensitivity and gauge factor. The thickness of the microcantilever should be small for achieving higher surface stress sensitivity. The individual thickness of each layer of the stack should be controlled to have the piezoresistive layer away from the neutral axis so that maximum surface stress can be transferred to the piezo layer. The high strain leads to large change in delta R resulting in higher gauge factors. The microcantilevers used in this work were fabricated using bulk micromachining. The microcantilevers have an overall stack (Silicon nitride-doped polysilicon-Silicon nitride) thickness of 900 nm, a length of 250 µm and width of 110 µm while having a spring constant of 0.2 N/m. The fabrication process started with an RCA cleaned double side polished (DSP) silicon substrate. SiO2 was deposited on both sides of the substrate. Bottom layer of SiO2 acted as a mask for etching silicon and top layer of SiO2, as an etch stop layer while etching silicon. A stack of silicon nitride, doped polysilicon and silicon nitride were deposited and patterned one by one. The bottom structural layer was made of 700 nm thick silicon nitride, deposited by Low Pressure Chemical Vapour Deposition (LPCVD) process and patterned. A piezoresistive layer of boron doped polysilicon (100 nm) was deposited and patterned. The top encapsulation layer is also of silicon nitride of thickness 100 nm. Windows were opened in the top silicon nitride, to create contacts to piezoresistor. Metal was deposited and patterned. The back side silicon oxide was patterned and silicon was etched using deep reactive ion etching (DRIE) process to form the pit so that the microcantilevers could be suspended. The top oxide was later etched using buffered hydrofluoric acid (BHF). The fabrication steps and SEM image of the released microcantilever is shown in fig. 2. We have also designed and fabricated a cost effective piezoresistive silicon nitride microcantilever platform with polymer anchor based on SPARED (Spin, Pattern, Anchor and Release Devices) MEMS process [13]. This process combines the advantage of surface micromachining of polymer with the material stability of silicon nitride (fig. 3). The device was characterized for all electrical, mechanical and electro-mechanical parameters. The microcantilever structure has a total stack thickness of 1075 nm with a spring constant of 0.9 N/m, deflection sensitivity of 0.3 ppm nm-1 and a gauge factor of 22. The resonant frequency and quality factor of this device is 22.5 KHz and 28 respectively as measured in air [14].
Fig. 3 (1) Fabrication sequence of piezoresistive nitride microcantilever with polymer anchor (a) substrate with sacrificial layer (b) structural layer silicon nitride of thickness 650 nm (c) patterned structural layer (d) patterned p-polysilicon on structural layer (e) patterned encapsulation nitride layer with contact window (f) patterned titanium /gold contact pads (g) polymer anchor (h) released microcantilever die (i) Optical image of a released die of silicon nitride microcantilever with polymer anchor (j) optical image of a released microcantilever die (2) (a) Plot of force-deflection curve from nanoindentation (b) Resonant frequency plot (c) Plot of ΔR/R as a function of deflection
III. P R IN C IP LE O F S E N SIN G AN D M ETH O D O LO G Y A microcantilever based platform having array of sensors is capable of determining several analytes, such as soil macronutrients and pH simultaneously. Polymeric ionophore doped ion-selective membranes were created using Polyvinylchloride-Dioctylsebacate (PVC-DOS) matrix which acts as a hydrophobic phase. Plasticizer like DOS is used to help in easy diffusion of ionophore in chelating the ions. It has been reported that this PVC-DOS mixture creates a homogeneous hydrophobic medium to facilitate the diffusion of ionophore, ion complexes similar to water-immiscible organic solvent. This polymeric matrix also improves mechanical stability [2]. To coat the microcantilevers with the ionophores, it is required that the ionophores are embedded in a polymer matrix. The analyte salt solution will remove the coatings of ionophore in absence of polymer matrix. In all our experiments, the membranes were prepared with the mixture consisting of 4 wt% ionophore, 66 wt% DOS and 33 wt% PVC. Since the microcantilever surface needs to be functionalized with this mixture, the thickness of the film is optimized by diluting it with tetrahydrofuran (THF). The porosity and roughness of the film were characterized by Atomic Force Microscope (AFM). The AFM image of the optimized film is shown in fig. 4-1. The cross-section Scanning Electron Microscope (SEM) image of the film shows the thickness to be 25nm as shown in fig. 4-1(d). Ionophores in the polymer matrix of PVC and DOS were asymmetrically immobilized on the microcantilever surface for lab-on-chip-detection of soil macronutrients NPK. Sensor array can also be helpful in improving the selectivity by having multiple sensors coated with different receptor analytes and decision can be made on sensing using a pattern recognition algorithm. Given below are the specific ionophores used for NPK detection. Potassium Sensing: Ionophores like Valinomycin [4] and 18-crown-6 ether [15], [16] have been reported in the literature for potassium sensing. ISE and ISFETs have used Valinomycin based PVC and plasticizer membranes for potassium ion sensing. Here we report use of 18-crown-6 ether as potassium ionophore for our piezoresistive micocantilever based sensing experiments. The schematic representation of the coating and the host guest interaction of crown ether with the potassium ion on microcantilever surface is shown in fig. 4-2 (c & d). Nitrate Sensing: Similarly, for nitrate sensing, we have used methyltridodecylammonium nitrate (MTDAN) and nitrate ionophore VI in polymer matrix [17]. Phosphate Sensing: Sasaki et al. have shown that the ion selective membrane containing tributyltin chloride as the ionophore exhibited high selectivity for H2PO4 with the addition of 25 mol% sodium tetrakis-[3,5- bis(trifluoromethyl)phenyl]borate (NaTFPB) [18]. For lab-on-a-chip detection of phosphates, we have used the same composition of ionophore and additive in the polymer matrix.
Fig. 4 (1) 3D AFM micograph of a drop casted surface showing good porosity which is required for diffusion of ions into the film for sensing purpose (b) 2D micrograph of the film (c) plot showing the height profile versus scanned distance (d) Cross section Scanning Electron Microscope (SEM) image of the film (thickness 26 nm), bottom silicon dioxide thickness 110 nm (2) (a) Molecular representation of 18-crown-6 ether (b) Molecular representation of 18-crown-6 ether with potassium ion in the cavity (c) Schematic representation of 18-crown-6 ether immobilized on microcantilever surface in a PVC/DOS matix (d) Schematic representation of potassium ion trapped in crown ether cavity resulting in deflection of microcantilever
IV. E X PE R IM EN TA L SE T - U P FO R LAB - O N - A - C H IP S O IL N U T R IEN T AN A LY S IS A microcantilever die consists of an array of four microcantilevers as shown in fig. 5 (a). This was mounted on a printed circuit board (PCB) and contacts were made using either wire bonding or silver epoxy. A Teflon flow cell with inlet and outlet was placed on the PCB. Out of four microcantilevers, one was used as a reference and the other three were test cantilevers used for sensing. The test microcantilevers are coated with receptor molecules embedded in a polymer matrix of PVC and DOS. The reference microcantilever is coated with only PVC and DOS to nullify the effect of soil solution on PVC/DOS polymer matrix. The surface of the microcantilever is asymmetrically immobilized with the polymer matrix using either a nano-dispenser or micropipette as shown in fig. 5 (b). The asymmetric coating of microcantilever is shown in fig. 5 (e & f). Fig 5 (e) shows the top side of the microcantilever coated with a PVC/ DOS based matrix along with a specific ionophore. A complete experimental set up is shown in fig. 5 (d). Microcantilever mounted on a PCB which is connected to a four channel resistance measurement setup is shown in fig. 5 (f). Once the devices are coated with ionophore embedded polymer, cured and packaged, these cartridges can be used for few months without any problem. Once the devices are used, they can’t be reused.
Fig. 5 (a) A microcantilever die (b) Coating an individual microcantilever with the help of a nano dispenser (c) microcantilever mounted in flow cell (d) complete experimental set-up (e) microcantilever coated with a PVC/ DOS based matrix along with a specific ionophore demarcating the extent to which the microcantilever is coated (f) microcantilever when viewed on the reverse side (g) A PCB connected to a four channel measurement system
Initial focus was to sense individual analytes with different ionophores. Further, the experiments were extended to sense multiple analytes and also study the effect of interfering ions. The experiments were carried out with two different methodologies. In the first method, 100 µl of 1 mM KCl solution was introduced at a constant flow rate using a syringe pump. In the second case, 100 µl of DI water was introduced into the flow cell by using a micropipette. The introduction of fluid causes a jump in resistance. Once the resistance values are steady in fluid, 10 µl of known concentration of KCl solution was introduced in the flowcell. 10 µl of sodium nitrate solution was introduced for nitrate sensing. V. R ESU LT S AN D D IS C U SS IO N Ionophores, named earlier as ion complexes are basically charge carriers or charge bearers. These molecules have a hydrophilic interior which binds specific ions and a hydrophobic exterior that interacts with the hydrocarbon interior of the membrane. Most ionophores form a cyclic ring concentrating oxygen and nitrogen functional groups at the center of their structure to associate with the cation or anion. The size of the cavity determines which ion can be encapsulated [19]. The six oxygen atoms of the 18-crown-6 ether interact with the bound K+ ion and these interactions are purely electrostatic. The sodium ions are smaller in size and they cannot interact at a time with six oxygen atoms and thus are not energetically favorable. The concentration gradient between organic film and aqueous phase helps the diffusion of the analyte ions through the film. The complexation of an ion in an ionophore results in conformal changes which in turn generates the stress on the microcantilever surface. Also since ionophores are charge bearers, the complexation of ion in ionophore results in charge separation layer of few nanometers. Since the microcantilevers are asymmetrically coated, it leads to accumulation of charges on one side and the conformal changes of ionophore results in the deflection of the microcantilever [20], [21]. The schematic of the same is depicted in fig. 6 (a). The deflection of the microcantilever generates the strain in the piezoresistive layer of the microcantilever resulting in change of resistance. Changes in resistances of all the microcantilevers were recorded using a four channel measurement system. The block diagram of the schematic of the measurement setup is shown in fig. 6 (b).
Fig. 6 (a) Schematic diagram depicting the charge accumulation and double layer formation on one side of the surface resulting in deflection (b) Block diagram of the portable system for point of use agriculture application
The plots of fig. 7 (a), 7 (b) and 7 (c) show the results of potassium sensing using 18-crown-6 ether. In fig. 7 (a), 1 mM of KCl was introduced into the flow cell at 6th min and the change in resistance was plotted with respect to time. In case of fig. 7 (b) and (c), after the resistance achieves a steady value in DI water, a known amount and known concentration of KCl is added resulting in 1 pM effective concentration of KCl solution in first case and 45 µM in second case.
Fig. 7 Sensor response to asymmetrically coated microcantilever with 18-crown-6 ether to potassium ions
Two different ionophores, nitrate ionophore VI and MTDAN were used for nitrate sensing. The fig. 8 (a & b) shows the response to nitrate ion analytes. In 8 (a), nitrate solution was introduced after the microcantilevers were stable in DI water and for 8 (b), the nitrate solution was introduced in the beginning itself. The microcantilever which was coated with nitrate ionophore showed the response to the analyte salt solution whereas the reference microcantilever which was just coated with polymer matrix did not show any response.
Fig. 8 Plots of nitrate sensing (a) Sensor response of microcantilever asymmetrically coated with nitrate ionophore VI in polymer matrix to nitrate ions (b) Sensor response of microcantilever asymmetrically coated with MTDAN in polymer matrix to nitrate ions.
Control experiments were carried out to confirm the selectivity and sensitivity of the ionophores. In the first set of experiments, the test microcantilever was functionalized with 18-crown-6 ether in a polymer matrix as described previously. Once the microcantilevers were stable in DI water, a known amount and concentration of sodium nitrate was introduced in the flowcell in the 13th minute. As seen in fig. 9 (a) the microcantilevers coated with 18-crown-6 ether did not show any response to the nitrate ions. In the second experiment, the test microcantilever was functionalized with MTDAN in a polymer matrix. A known amount and concentration of KCl was introduced in the flowcell in the 12th min after stabilization of the microcantilevers. As seen in fig. 9 (b) the microcantilevers coated with MTDAN did not show any response to the potassium ions. This shows that the ionophores are highly selective to particular ions.
Fig. 9 Plots of ionophore subjected to interfering ions (a) Sensor response of microcantilever asymmetrically coated with 18-crown-6 ether (potassium ionophore) in polymer matrix to nitrate ions (b) Sensor response of microcantilever asymmetrically coated with MTDAN (nitrate ionophore) in polymer matrix to potassium ions.
V I. C O N C LU S IO N As part of this work, we have demonstrated for the first time the measurement of soil macronutrients (NPK) using MEMS based lab-on-a-chip system. Sensing of potassium and nitrates has been demonstrated using 18-crown-6 ether, MTDAN and nitrate ionophore VI embedded in a polymer matrix. Similarly, we could successfully create and asymmetrically coat phosphate selective ionophore polymeric matrix on to the microcantilever. Currently, we are working on the phosphate sensing experiments. This platform can be further extended to test various other soil parameters like soil micronutrients and pathogens. A low-cost silicon nitride microcantilever platform is also developed for the agricultural applications. VII. A C K N O WLED G M EN T This work was supported by the Department of Science and Technology, Department of Electronics and Information Technology, Government of India, through the Centre of Excellence in Nanoelectronics. We would like to thank Dr. Madhuri Vinchurkar for the discussions.
R E FE R EN C ES [1] [2] [3] [4]
H.J. Kim et al., “Soil macronutrient sensing for precision agriculture,” J. Environ. Monit, vol. 11, pp. 1810–1824, 2009 Philippe Buhlmann and Li D. Chen, “Ion-Selective Electrodes With Ionophore- Doped Sensing Membranes”, In Supramolecular Chemistry: From Molecules to Nanomaterials, John Wiley & Sons, , 2012 Akshay Sankpal and Krishna K. Warhade, “Review of Optoelectronic Detection Methods for the Analysis of Soil Nutrients”, International Journal of Advanced Computing and Electronics Technology, Vol. 2, No. 2, 2394-3416, 2015 H.J Kim et al, “ Evaluation of nitrate and potassium ion-selective membranes for soil macronutrient sensing”, Transactions of the ASABE, Vol. 49(3), pp 597-606, 2006
[5] [6] [7] [8] [9] [10] [11] [12] [13]
[14] [15] [16] [17] [18] [19] [20] [21]
J. R. Brown and R.R Rodriguez, “ Soil Testing in Missouri- A Guide for conducting soil tests in Missouri”, University of Missouri, Extension Circular No 923, 1983 Nickolay V. Lavriket et al., “Cantilever transducers as a platform for chemical and biological sensors”, Review of scientific instruments, Vol. 75, No. 7, pp. 2229-2253, 2004 R. S. Patkar, M. G. Seelan, and V. R. Rao, “A highly sensitive piezoresistive cantilever based sensor platform for detection of macronutrients in soil,” in Proc. IEEE- NANO, pp 751-754, Jul 2015. Anja Boisen et al, “ Cantilever like Micromechanical Sensors”, Rep. Prog. Phys, Vol. 74, pp30, 2011. Sen Xu and Raj Mutharasan, “ Cantilever Biosensors in drug discovery”, Informa Healthcare, Vol.4, No 12, pp1237-1251, 2009. Larry Sensac and Thomas Thundat, “Nanosensors for explosive detection”, Materials Today, Vol.11, No. 3, pp 28-36, March 2008. V. Seena et al, "Polymer Nanocomposite nanomechanical cantilever sensors: Material characterrization, device development and application in explosive vapour detection”, Nanotechnology, Vol.22, No. 29, pp 11, 2011. S. M. Ali et al, “Mechanical Performance of cantilevers in liquids”, J. Microelectromech. Syst., Vol 20, No.2, pp 441-45, April 2011. Rajul S. Patkar, et.al., “Polymer-Based Micro/Nano Cantilever Electro-Mechanical Sensor Systems for Bio/Chemical Sensing Applications” Micro and Smart Devices and Systems, Editors: K.J. Vinoy, G.K. Ananthsuresh, Rudra Pratap, S.B. Krupanidhi, Springer Tracts in Mechanical Engineering 2014, pp 403-422, ISBN 978-81-322-1912-5(Print) 978-81-322-1913-2(online) Rajul S Patkar et al., “ A novel SU8 polymer anchored low temperature HWCVD nitride polysilicon piezoresistive cantilever”, IEEE J-MEMS, Vol. 23, No.6, December 2014 Mohammed Reza Ganjali et al, “Supramolecular Based Membrane Sensors”, Sensors, Vol. 6, No.8, pp1018-1086, 2006. Chang Min Choi et al., “Binding selectivity of dibenzo-18-crown-6 for alkali metal cations in aqueous solution: A density functional theoy study using a continuum solvation model”, Chem. Cent. J, vol. 6, No. 84, pp. 8, 2012. J. Gallardo et al, “A flow-injection electronic tongue based on potentiometric sensors for the determination of nitrate in the presence of chloride”, Sensors and Actuators B: Chemical, Vol. 101, No.1, pp 72-80, 2004 S. Sasaki et al, “ Organic Tin Compounds combined with anionic additives- an ionophore system leading to a phosphate ion selective electrode” Talanta, Vol. 63, pp 131-134, 2004 Manihiro Hillary, “Mechanism of action of ionophores, examples and their significance in a cell”, Slideshare (http://www.slideshare.net/ManirihoHillary/ionophores), 2016 Neng-Hui Zhang et al, “Effect of Surface Charge State on the surface stress of a microcantilever”, Nanotechnology, 27, pp. 1-10, 2016 Thomas Thundat, United States Patent, Patent No: 6,016,686, Micromechanical Potentiometric Sensors, 2000