An optimized measurement chamber for cantilever array measurements in liquid incorporating an automated sample handling system
© Walther et al. 2015
Received: 17 November 2014
Accepted: 27 April 2015
Published: 12 May 2015
Micro- and nanomechanical analytical devices for diagnostics require small sample volumes, and stable and efficient clamping of the sensors for optimized bioassays. A fully automated device for the readout of the dynamic and static response of cantilevers in a physiological liquid environment including a sample handling system is presented. The device provides sequential readout of the static and dynamic mode providing the best signal to noise ratio for each individual cantilever. In the dynamic mode, it is possible to measure up to the 16th flexural resonance mode of vibration of a 500 μm long and 1 μm thick cantilever. The automated sample handling system enables the local injection of sub microliter volumes of sample with excellent reproducibility. Data demonstrating the response of the cantilevers to external stimuli highlight the sensitivity of the device and the importance of the use of reference cantilevers to decouple biologically relevant metrics from environmental effects. A specific biomolecular interaction of sensitised nanospheres on cantilever sensors with a mass resolution of ~50 picogram in liquid is shown.
PACS numbers: 07.10 Mechanical instruments, 87.85 Biosensors, 47.85. Fluidic
Cantilever technology has been used for a wide variety of applications including mass, force and biological sensing. Within the latter category, a large subset of sensing applications exists in the fields of proteomics [1-5], genomics [6-8] and microbiology [9-15], amongst others. A crucial component in achieving biologically relevant metrics in bio-sensing is the environment in which the test is performed. The mainstays in routine bio-sensing applications, such as ELISA and qPCR, are performed in physiological (liquid) environments. To bridge the gap between research and clinical laboratories, cantilever sensors must follow suit despite the challenges encountered when operating in fluid. Operating within a relevant biological environment affords considerable validity to the technique in comparison to a ‘dip-dry’ method .
The handling of liquid volumes takes on an increasing relevance when moving from a ‘dip-dry’ method to a continuous measurement format, as liquid is transferred to the cantilever rather than the cantilever to the liquid. For efficient transfer of small volumes of liquids, the integration of micro-fluidic technologies, which already inevitably overlap with cantilevers in many fields, is desirable. Scaling down provides numerous advantages on both fronts; micro-cantilevers offer an increased sensitivity over their macro alternatives, and the conservation of precious samples in analytical techniques .
In order to de-couple environmental effects or spurious events from biologically relevant events, at least one reference cantilever should be used when utilising micro-cantilevers as bio-sensors . These environmental effects can further exacerbate when working in a liquid environment in comparison to a micro-cantilever operated in vacuo and include temperature, viscosity, and liquid flow. Without the appropriate in situ reference cantilevers, a measured signal cannot confidently be attributed to the investigated analyte property only [16,19].
The device presented here incorporates a micro-cantilever array operated in both static and dynamic mode in a liquid environment. Arrays allow reference cantilevers to be designated and the elimination of environmental influences. Sub microliter sample volumes can be injected easily and the device can be operated in stop flow or in continuous flow mode. The read-out principle of the nanomechanical array is based on the optical beam deflection method . The entire device is fully automated from optical alignment to sample injection, readout and analysis.
Fluidic chamber assembly
The chamber body has two fluid inlets and two outlets. Extruded 1/16” Teflon tubing (Zeus, Orangeburg, USA) is connected at each flat-bottom port using M6 PEEK nuts and flangeless ferrules (Idex Corporation, Illinois, USA). The remaining port houses a micro-dispensing valve (The Lee Company, Connecticut, USA) fixed using a manifold mount such that the outlet nozzle is protruding directly into the flow stream. This configuration allows the implementation of additional inlet ports in a manifold configuration ‘up-stream’ of the first injection port without redesign of the core of the measurement chamber.
In order to obtain a wide range of resonance modes, a stack of piezoelectric actuators (EBL Products Inc., Connecticut, USA), assembled in-house with the size of the cantilever array body, is mounted below the array body using TorrSeal® (Agilent Technologies Ltd., Torino, Italy). The stack is composed of three piezoelectric crystal layers each 1.0 mm thick. Two 2x3 mm2 piezoelectric pieces (EBL Products INC., East Hartford, USA) and one 2x2 mm2 piece (EBL Products INC., East Hartford, USA) are sandwiched together using electrically conductive epoxy resin (Epoxy Technology, Inc. Billerica, USA). The actuator is separated from the liquid chamber by a 200 μm thick PEEK membrane to prevent shorting. The membrane is sufficiently thin to allow adequate transfer of energy from the actuator to the cantilever (see Figure 2).
Adjacent to the piezoelectric actuator, and directly underneath the cantilever array, is situated a peltier module (Global Component Sourcing, Hong Kong). This module allows a heat pulse to be applied to the liquid chamber enabling a subsequent normalization of the nanomechanical response of the individual cantilevers to compensate for differences of their mechanical properties. A precision temperature sensor (Hygrosens Instruments GmbH, Löffingen, Germany) is placed in close proximity to the cantilevers in the PEEK chamber.
Liquid is introduced to the chamber using two different methods. A Genie™ Plus Syringe Pump (Kent Scientific Corporation, Connecticut, USA) is used to pump buffer through the chamber. The micro dispensing valve is used to introduce the sample of interest to the liquid line and is pressure driven. A compressed air regulator (Spectron Gas Control Systems GmbH, Frankfurt, Germany) provides the appropriate pressure in the 20 – 100 mbar regime and the desired volume is injected through controlling the opening time of the micro dispensing valve.
A three-way solenoid valve (ASCO Valve, Inc. Florham Park, NJ, USA) allows control over whether the liquid flows into the measurement chamber or whether it bypasses it using the priming outlet. The priming outlet is located between the measurement chamber and the two inlet ports. This outlet allows diverting bubbles and priming of the micro dispensing valve without introducing sample into the cantilever chamber before it is required.
Because the cantilever sensors are often coated on one side with one or more metal layers for subsequent chemical or biological functionalisation, care must be given to ensure a steady temperature. Fluctuations in temperature will result in the cantilever deflecting due to the difference in the thermal expansion coefficients of the silicon and the metal layers. Thermal fluctuations affect the viscosity and density of the liquid and therefore the oscillation frequency of the sensors. To ensure a steady temperature, the fluid chamber, the buffers, samples and peripheral equipment such as optics are all housed within a small refrigeration/heating unit (Intertronic, Interdiscount, Switzerland). A silent, vibration free fan (Zalman, Seoul, Korea) helped maintain a uniform temperature within the refrigeration unit.
Optical beam deflection system
Optical read-out is the most commonly used read out system and can give sub-angstrom resolution [18,20]. A single wavelength fibre-coupled laser diode (830 nm, 10 mW, C Pin Code, SM Fibre Pigtailed Laser Diode; LPS-830-FC; Thorlabs Ltd. Cambridgeshire, UK) was used. The laser has a free space power of 10 mW and a line width >200 kHz. Stabilisation within 1% is achieved once it has been turned on for 20 minutes. A combined laser diode and temperature controller (ITC4001 Benchtop Laser Diode/TEC Controller, 1 A/96 W; Thorlabs Ltd. Cambridgeshire, UK) allowed for a very stable output. The laser was coupled to a collimator (F260APC-780; Thorlabs Ltd. Cambridgeshire, UK) which collimated the beam to a 3.33 mm diameter. The laser beam is focused to a 14 μm spot on the cantilever using a 50 mm focal length achromatic doublet (AC127-050-B; Thorlabs Ltd. Cambridgeshire, UK).
The reflected laser spot is tracked using a linear position sensitive device (PSD 1 L10-10-A_SU15; SiTek Electro Optics AB, Partille, Sweden) with an in-house designed electric amplification circuit with a cut off at 2 MHz (3 dB), which extracts the differential and summation signal at a gain of 1. To avoid saturation of the PSD, a neutral density filter (NE506B-B; Thorlabs Ltd. Cambridgeshire, UK) can be inserted into the beam path to attenuate the laser.
Optical read-out of all eight cantilevers is possible by mounting the laser assembly within an optical cage system (Thorlabs Ltd. Cambridgeshire, UK) on high precision automated translation stages (122.2DD and M-110.1DG Precision Micro-Translation Stage; Physik Instrumente Ltd., Bedford, England). Aligning the stages to enable both x-axis and y-axis motion allows movement between cantilevers, and also adjustment to an optimum location along each cantilever. Particularly within dynamic mode operation, optimal amplitude signals can be obtained by individually positioning the laser on each cantilever separately, as this can compensate for subtle mechanical differences between the cantilevers. For the dynamic mode operation the laser has to be positioned and focussed on a flex point of the cantilever. For example, the node-to-node distance for the 16th flexural resonance mode is ~33 μm, hence the angle sensitive region is approximately 11 μm long. The y-axis and x-axis miniature translation stages feature an optical linear encoder with 100 nm and 50 nm position resolution and a velocity of 20 mm/s and 1 mm/s respectively.
The device outlined can be operated in both dynamic and static mode. When set to dual mode operation, the laser spot cycles through the cantilevers sequentially, taking firstly a dynamic measurement and then a static measurement before moving onto the next cantilever. A LabVIEW (National Instruments, Texas, USA) program controls the movement of stages, dictates the frequencies sent to the piezoelectric actuator and performs analysis of the signals acquired by the PSD. LabVIEW is also used for temperature and fluidic control.
Automated laser focusing
To obtain the highest resolution possible for the static measurements, the laser has to be well focussed and positioned at the apex of the cantilevers where the maximum deflection occurs . To acquire the maximum response for all the flexural modes of vibrations of a cantilever operated in dynamic mode, the laser has to be precisely positioned and focussed at a node of the cantilever . Therefore, the widely used knife-edge technique [23-25] to determine the laser beam width is embedded in the LabVIEW code to perform the focusing of the laser on the cantilever automatically.
The laser is moved onto the cantilever where it is reflected onto the PSD. As the laser is then moved across the cantilever, the intensity of the reflected light is measured by the PSD and recorded as a function of its position on the cantilever. By moving the laser off from the cantilever, less light is reflected and the measured sum signal continuously decreases to zero.
The LabVIEW program iteratively changes the laser distance with respect to the cantilever and calculates the beam radius until a laser spot radius of less than 8 μm is achieved. This quick and inexpensive method gives an accurate determination of the laser spot radius, resulting in an optimally focused laser on the cantilever.
Dynamic mode measurement
Static mode measurement
Silicon cantilever arrays (IBM Research Laboratory, Rüschlikon, Switzerland) with eight cantilevers per array were used for all measurements. The cantilevers had a thickness of 1 μm, a length of 500 μm and a width of 100 μm. The cantilevers were designed with a 250 μm pitch and cantilevers from the same wafer possessing the same dimensions were used for all experiments.
Automated laser focusing
The laser was moved onto the initial position on the cantilever where it was reflected onto the PSD. As the laser was then moved across the cantilever, the intensity of the reflected light was measured by the PSD and recorded as a function of its position on the cantilever. The model from De Araújo  was fitted to the experimental data and the radius of the laser beam on the cantilever surface was obtained. Based on the obtained radius, the optical cage was moved closer or further away from the measurement chamber followed by the next measurement to obtain the radius of the newly focused laser beam. This process was repeated until the laser was finely focused on the cantilever. The depth of focus of the laser of several tens of microns allowed for variation in the position along the x-axis without compromising the focussing of the laser.
Thermal noise versus active dynamic actuation with the integrated piezo
A cantilever array was loaded into the fluid chamber and allowed to equilibrate in deionized water. For the thermal noise power spectrum of the sensors a frequency domain from 0 - 18 kHz was analysed with a sampling rate of 90 kHz and a frequency resolution of 0.1 Hz. The parameters for the power spectrum analysis were: a Hamming window  with 200 rms averages. The data can be further smoothed without loosing data information applying a Savitzky-Golay algorithm . Within this range the first, second and third harmonic of the 1 μm thick cantilevers in liquid could be investigated. The voltage signal of the differential thermal noise on the PSD could be amplified by factors up to 5000 without driving the sensors with the integrated piezo-electric stack. Alternatively the sensors were then actively driven with the piezo-electric stack by applying an actuation voltage within the range of 2 – 5 volts through the software.
A cantilever array was loaded into the fluid chamber and allowed to equilibrate in nanopure water. When the temperature reached steady state at 23.1°C, dual mode measurement was started. Every two hours, the set-point temperature was increased by 0.5°C until a temperature of 24.1°C was reached. The temperature stability at the set point is ±0.02°C.
The dual mode sweep occurred every 30 seconds, upon which dynamic mode measurements, static mode deflection and temperature were recorded. For the dynamic mode operation, the range for the frequency sweep was set from 200 kHz to 500 kHz within which three resonance mode peaks were contained . To ensure that only bimetallic effects of the sensors contribute to the evaluated static cantilever signal a chip with solid sidebars in position 0 and 9 was scanned. These additional signals showed that the drift of the device is neglectable .
Flow rate effects
A cantilever array was loaded into the fluid chamber and allowed to equilibrate in nanopure water. When the temperature reached steady state at 23°C, dual mode measurement was started. The flow rate was set to 2 μl/min, 5 μl/min, 10 μl/min, 25 μl/min, 50 μl/min and 100 μl/min sequentially for 30 minutes each. The buffer reservoir was within the temperature controlled unit to ensure a uniform temperature throughout the experiment.
The dual mode sweep occurred every 30 seconds, upon which dynamic mode measurements and temperature were recorded. For the dynamic mode operation, the range for the frequency sweep was set from 250 kHz to 400 kHz within which two resonant mode peaks were contained.
Sealing of the Chamber
The influence of O-ring or Nescofilm sealing on the static mode signal was investigated. A cantilever array was loaded into the fluid chamber that was subsequently sealed either with Nescofilm or an O-ring and allowed to equilibrate in nanopure water. After equilibration, the static mode measurement was started. Nanopure water was introduced into the chamber at a flow rate of 10 μl/min for 45 minutes or in a second experiment 2 μl/min for 5 minutes.
Volume of injected samples
A 100 μl volume of nanopure water was loaded into the sample reservoir and the injection valve was primed. A series of injections at a constant pressure of 100 mbar were performed with increasing valve opening times from 31 ms to 40 ms in 1 ms increments. The sample injection line was secured along a graduated surface and the sample meniscus was monitored after each injection to determine the volume of fluid injected. A digital microscope (Dino-Lite Pro HR AM7000 series; Dino-Lite, The Netherlands) was used to give the required resolution. To determine the volume injected, the pixel location of the meniscus along the graduated scale was determined using images from before and after injection. This pixel differential was then converted to length, and hence volume (ranging from 4 to 5 μl), using the ratio of millimetres to pixels which could be easily calculated using image analysis software.
A series of ten 500 nl samples were also injected to investigate the reproducibility of the injected volumes and were analysed using the same method as above.
A cantilever array was loaded into the measurement chamber and allowed to equilibrate in 5% glycerol solution (v/v). When the temperature reached steady state at 23°C, dual mode measurement was started. After 15 minutes, a 10 μl volume of 12% ethylene glycol (v/v) was injected, which is enough to exchange the liquid in the fluid chamber. After a further 30 minutes, the measurement chamber was flushed with 100 μl of 5% glycerol solution at a flow rate of 20 μl/min. The 5% glycerol solution has a theoretical viscosity of 1.055 cP while the 12% ethylene glycol’s viscosity is 1.257 cP at 20°C.
The sweep occurred every 15 seconds, upon which dynamic mode measurement and temperature were recorded. For the dynamic mode operation, a frequency sweep between 250 kHz and 600 kHz was generated within which three resonance mode peaks were tracked.
A viscosity change from water (1.0 cP at 20°C) to 12% ethylene glycol (v/v) (1.257 cP at 20°C) was induced to analyse the benefit from an actively versus thermal noise driven system. For comparison the same viscosity change from water to ethylene glycol solution at mode 13 was investigated. The thermal noise peaks were fitted using a Lorentzian fit to enhance the visibility of the resonance peaks and to determine the resonance frequency.
A cantilever array was loaded into the measurement chamber and allowed to equilibrate in 5% glycerol solution (v/v). When the temperature reached steady state at 23°C, dynamic mode measurement was started. After 15 minutes, a 10 μl volume of 5% ethylene glycol (v/v) was injected. After a further 30 minutes, the measurement chamber was flushed with 100 μl of 5% glycerol solution at a flow rate of 20 μl/min. The 5% glycerol solution has a theoretical density of 1010 kg/m3 while the 5% ethylene glycol’s density is 1003 kg/m3 at 20°C.
The sweep occurred every 15 seconds, upon which dynamic mode measurement and temperature were recorded. For the dynamic mode operation, a frequency sweep between 200 kHz and 550 kHz was generated within which three resonance-mode peaks were tracked.
To compare the performance of the new fluidics and measurement chamber to previous experiments a biomolecular recognition proof of concept experiment for biological sensing was implemented. A chip with 8 gold coated sensors was functionalised with ω-terminated alkyl-thiols to form self-assembled monolayers . Four sensors were functionalised with a hydroxy-terminated functional group (H338, Dojindo, Munich, Germany) and four with a biotin-terminated group (B564, Dojindo, Munich, Germany) in a 1 mM concentrated ethanol solution in a micro-capillary functionalisation tool  for 30 minutes at room temperature. After the functionalisation the sensors were thoroughly rinsed with first ethanol, water and then with PBS buffer (pH 7.4).
As a target for biomolecular specific interactions polystyrene spheres (diameter 810 nm, 3.42 x 1010 beads/ml, SVP-08-10 Spherotech, Lake Forest, IL) functionalised with streptavidin were utilised. 10 μl of beads were washed by re-suspending the sedimented pellet three times with 1 ml PBS after centrifugation for 5 minutes at 5000 rpm. Finally the beads were resuspended at a concentration of 3.42 × 108 beads per μl.
After initial equilibration of the sensors in PBS buffer with 10 μl/min flow at 24°C for five minutes the buffer was stopped for 13 minutes. During this wait time the sample volume of 10 μl streptavidin beads was loaded upstream of the measurement chamber with the micro-dispensing valve into the liquid channel by opening the priming channel. Then the sample was pushed into the liquid chamber with a speed of 10 μl/min for two minutes to ensure full exchange. The flow was then stopped for 10 minutes to facilitate a specific interaction of the streptavidin spheres with the biotin – or hydroxyl functionalised sensors. To conclude the experiment the chamber was rinsed for 20 minutes with PBS buffer at 10 μl/min. During the whole biological assay two resonance peaks per sensor (mode 10 and 11) with 3000 data points per peak were measured with a sampling rate of 5 × 106 samples/s. Each data point is the average of 5x103 samples. To plot the data, the signals of four biotinylated and four of the hydroxyl functionalised cantilevers were individually averaged. The frequency shifts measured were converted to mass changes using the formulas given in ref .
Results and Discussion
Automated laser focusing
Thermal — versus active dynamic actuation
In Figure 8 we report on the quality of the micromechanical resonance spectra of the cantilevers within the array applying an actuation voltage on the piezo-electric stack mounted underneath the cantilever array. The frequency response of the piezo-electric stack was non-linear and showed an increased performance towards higher frequencies . By driving the piezo-electric stack up to 4 - 5 volts we generated optimal detectable resonance peaks at higher modes. The actuation settings normally used for data acquisition were around \( \pm \) 4 volts (range \( \pm \) 0 - 5 volts) and a gain of 500. The resonance peaks at higher modes could be easily visualised (up to mode 16 in this publication) whereas in a situation when only thermal ‘noise’ would be used, natural frequencies higher than mode 2 would not be recognisable. The differential signal contribution for specifically measured interactions that change the mass of the sensors scale proportionally with higher modes. Therefore the implementation of a piezo-electric stack with the size of the cantilever array mounted directly underneath the sensor chip was highly beneficial to boost the sensitivity of the method .
Sealing of the chamber
Volume of injected samples
While Figure 14A shows slight variation around the trend line, it does not give an indication of the reproducibility of the injection volume using set parameters. A series of ten 500 nl volumes (at 100 mbar) were injected and their volume plotted against droplet number (shown in Figure 14B). Excellent reproducibility was demonstrated as the coefficient of variation (CV) for this series of droplets was 1.3%. A video highlighting the injection and exchange of the solution in the measurement compartment is shown in (Additional file 1).
A completely automated device for readout of the dynamic and static response of cantilevers in a physiological environment is presented. The device enabled the local injection of sub microliter volumes of sample with excellent repeatability. Reducing the sample volume from previously hundreds of μl [22,41] to a few μl enables the ability to generate larger volumes of data from the same total sample volume and hence the conservation of precious samples. We investigated the effect of the chamber seal on the nanomechanical measurement with either a thin membrane or a conventional O-ring. Both sealing methods are valuable for all liquid experiments and create a 4 μl measurement chamber which requires approximately 10 μl of liquid for full exchange. A slow injection speed (2 μl/min) is recommended in static mode measurements to minimise hydrodynamic bending of the sensors. The complete automation of the device including laser focusing allows the greatest resolution possible from experiment to experiment and reduces error due to user interactions. A series of experiments demonstrating the response of the cantilevers to external stimuli (temperature, viscosity, density, flow rate) highlight the sensitivity of the device. The difficulty of deconvoluting unambiguously relevant biological events in measurements using a single nanomechanical sensor is highlighted when one considers any sample introduced to the sensor may have a slight variation in temperature/viscosity/refractive index to the buffer fluid. It also serves to emphasise the importance of the use of in situ reference cantilevers to decouple biologically relevant metrics from environmental effects in real-time as shown when cantilevers are used as specific biosensors.
This work was supported by Science Foundation Ireland under the CSETscheme SFI08/CE/I1432, PI scheme SFI/09IN/1B2623 and SFI12/TIDA/B2380.
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