A microfluidic AFM cantilever based dispensing and aspiration platform
© van Oorschot et al.; licensee Springer. 2016
Received: 17 June 2014
Accepted: 24 December 2014
Published: 12 March 2015
The Erratum to this article has been published in EPJ Techniques and Instrumentation 2016 3:4
We present the development of a microfluidic AFM (atomic force microscope) cantilever-based platform to enable the local dispensing and aspiration of liquid with volumes in the pico-to-femtoliter range. The platform consists of a basic AFM measurement system, microfluidic AFM chip, fluidic interface, automated substrate alignment, external pressure control system and controlled climate near the dispensing area. The microfluidic AFM chip has a hollow silicon dioxide (SiO2) cantilever connected to an on-chip fluid reservoir at one end and a slicon nitride (Si3N4) tip with an aperture on the other end. A 3D printed plastic fluidic interface glued over the on-chip reservoir was used to connect microfluidics and macrofluidics. The fluidics is connected to an external pressure control system ranging from −0.8 bar to 5 bar with 0.1 bar resolution. This pressure range allows dispensing and aspiration of liquids through the cantilever tip aperture. The controlled climate with a temperature control range between 25°C – 40°C and humidity up to 95% near the dispensing area keeps the droplets for sufficiently long time before they evaporate. An array of droplets can be programmed to be dispensed automatically and access them again with a position accuracy of 1 micron. Experiments were performed with two types of cantilevers with different geometrical configurations. A minimum flow rate control of 50 fL/s was obtained and also frequency shift was monitored as the cantilever was filled with liquid. This platform will be used for various chemical and biological applications.
The ability to controllably infuse or withdraw different types of reagents with nanometer precision unlocks the door for new processes that can lead to novel opportunities like cell surgery. Lately, more applications, particularly in chemistry and biology are continuously demanding the development of certain tools that would allow the precise dispensing and aspiration of extremely small amounts of liquids at predefined positions. In on-going efforts to achieve this, different types of fluid dispensing devices, such as micropipettes and ink-jet printers, have been explored. Nevertheless, these devices do not offer the degree of precision and control that is required to explore the intended applications. Furthermore, they are limited to handle volumes in the order of micro-to-picoliter [1,2], which constitutes at least three orders of magnitude bigger than the suggested (sub)femtoliter regime for most of the upcoming applications. In attempts to manipulate liquid in the smallest possible regime, scientists have started to develop cantilever-based techniques (i.e. DPN , NADIS , NanoFountain Probe  and FluidFM ) which have been proved to surpass the picoliter barrier and handle volumes as small as few zeptoliters . These devices, compatible with Atomic Force Microscopy (AFM), combine the versatility of microfluidics with the high precision provided by the cantilever . The challenge, nevertheless, remains on achieving controlled dispensing of the desired volume despite the complications given by external factors such as evaporation , humidity , hydrophobicity of the tip , surface energy of the substrate  and viscosity of the liquid . Additionally, the incapability to aspirate controlled amounts of liquid has slowed down the potential break-through applications. In order to overcome these challenges and get closer towards the envisioned goals, we have developed a microcantilever AFM-based platform to enable the dispensing and aspiration of liquids with volumes in the femtoliter range under controlled environment. The various parts in this system constitute a basic AFM measurement system, a microfluidic chip, an automated substrate alignment, a humidity chamber and a microfluidic interface connected to an external pressure control system. The microfluidic chip consisted of a hollow silicon nitride (Si3N4) tip and channelled silicon dioxide (SiO2) cantilever which were connected to a fluidic reservoir located in the handling part of the chip. After filling the reservoir with liquid, a positive or negative pressure relative to the ambient is applied in the reservoir to infuse or withdraw liquids through an aperture located at the apex of the tip. Constant volume systems such as a syringe pump used for applying pressure differences suffer from the effect of shock waves produced by the stepper motors and have a slow response at low flow rates. Moreover, it can take up to few minutes for the pressure to stabilize. Therefore, we have chosen the constant pressure system which is pulse free, has a fast response and can also produce high flow rates. The results shown here represent an improvement over our earlier work on thermal pumping , evaporation based pumping and aspiration  and syringe-pump based pipetting .
Results and discussion
Fluid filling and substrate stage
We found that when mounting and gluing the microfluidic chip to the plastic interface, which are both mountable in standard AFMs, the maximum pressure that can withstand before detaching was >5 bar. The system allowed to regulate the pressure between −0.8 bars and 5.0 bars with better than 0.1 bar resolution, while a valve accurately timed can switch between the regulator set point and ambient pressure within 100 ms.
The temperature and humidity control resulted in precise regulation of temperature between 25-40°C and humidity up to saturation point. Some condensation was noticed around saturation point at high humidity and temperature.
For type-A cantilevers the resonance frequency shift as the fluid was filled (Figure 2b) inside the hollow cantilever was noted. For each pressure increase step, the liquid meniscus advancement was noted, At 1.2 bar of applied external pressure, the entire cantilever was filled. At appropriate applied pressure, equilibrium was established between evaporation of the droplet at the tip and the supplied liquid.
Characteristics of the hollow cantilever and microfluidic cantilever-based platform
Cantilever dimensions (each leg)
Type-A: L:155 μm, W:42 μm, T:4.9 μm Type-B: L:155 μm, W:6.4 μm, T:4.9 μm
Channel dimensions (each leg)
Type-A: L:153.5 μm, W:40 μm, T: 2.2 μm Type-B: L:153.5 μm, W:3.7 μm, T: 2.2 μm
Type-A: 2.4 N/m (when empty); Type-B: 9.4 N/m (when empty)
Type-A: 153.94 kHz (when empty); Type-B: 110 kHz (when empty)
Aspiration and dispensing flow rate
50 fL/s @1.5 bars
0.85 bars under pressure to 5.0 bar overpressure; 0.01 bar resolution
Settling time at dispenser out
Ambient to 40°C ±0.5
30% – 90% RH ±5% non condensing 90% – 100% RH ±5% condensation
Between chip positions on the same substrate: <1 μm Between different chips: <2 μm
To conclude, we have developed a hollow AFM cantilever-based platform able to dispense and aspirate liquids in the picoliter to femtoliter volumes of liquid. Besides regular AFM measurements, the AFM chips and the system are designed to handle these liquid volumes in a controlled climate suitable for numerous chemical and biological applications.
Methods and materials
Microfluidic chip and fluidic interface
In order to connect the hollow cantilever to the pressure control system and avoid leakage during liquid transport, a microfluidic plastic interface was manufactured. SolidWorks Software was used to create a suitable model (Figure 3b), which was later printed out of HTM140 polymer using Objet 3D-printer. The polymer has good temperature resistance and high tensile strength.
Bonding the microfluidic chip to the interface
Microfluidic AFM dispensing and aspiration platform
External pressure control system
Temperature and humidity control
Substrate to tip alignment
Two Newport SMC100 stages with a range of 25 mm were mounted under the substrate (Figure 5a). These stages are used for XY positioning the substrate and the chip. Silicon oxide chips from Bioforce with a 100 μm square grid were used to align on. The edges of these gridlines were detected with machine vision and software determined the tip and substrate position. The software could position the tip in one of the squares and select a position within this square. This could be used to find back a previously dispensed droplet within an accuracy of 1 micron. After positioning, the AFM piezo stages could be used for sub-micron positioning.
Dispense automation software
This work is supported by NanoNextNL, a micro and nanotechnology consortium of the government of the Netherlands and 130 partners
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