Jeroen A van Kan*

Centre for Ion Beam Applications, Department of Physics, National University of Singapore, 117542, Singapore

* Email: phyjavk@nus.edu.sg; Tel.: +65-65166978

Proton Beam writing (PBW), a new 3D nanolithographic technique is used to produce smooth 3D nanostructures. In combination with UV lithography these structures form masters for soft lithography and nanoimprint lithography. We will demonstrate the successful reproduction of PMMA and PDMS LOC devices featuring details down to 60 nm and or high aspect ratios. These LOC devices are extensively used for DNA single molecule analysis and large scale genome sequencing [1]. A second application in the area of particle separation will also be discussed. Here active particle separation and sorting is achieved harnessing periodically switched magnetic fields and Brownian motion of micro size particles. Here the particle separation is achieved in a compact microfluidic chip with high aspect ratio asymmetric sawtooth sidewalls in the sorting channel. The magnetic field is generated via co-fabricated electromagnet metal solder channels [2].

PBW is an ideal technique to fabricate this type of lab-on-chip devices featuring smooth high aspect ratio structures at the micron as well as at the nano scale. PBW is a new direct write 3D nano lithographic technique which has been developed at the Centre for Ion Beam (CIBA), in the Physics Department of the National University of Singapore. PBW employs a focused MeV proton beam which is scanned in a predetermined pattern over a resist, which is subsequently chemically developed. PBW exhibits low proximity effects coupled with the straight trajectory and even energy deposition along the path of the proton beam results in sidewall smoothness of a few nm root mean square. The high penetration depth of the proton beam enables the production of high aspect ratio, high density 3D micro and nano structures with smooth sidewalls, ideal for high quality mold production for Nano Imprint Lithography (NIL) applications. The PBW system is now able to focus proton beams down to 9.3 x 32 nm² [3], allowing high aspect ratio lithography down to 19 nm in HSQ. To achieve polymer nanofluidic circuits down to 19 nm PWB fabricated HSQ resist structures will be replicated in OrmoStampTM (Micro Resist Technology GmbH) to form hard stamps which can be used in thermal NIL. Initial NIL results obtained with these molds are presented in combination with PMMA nanofluidic lab on chip production. To improve on the ultimate feature size and make the PBW system more user-friendly a tabletop PBW system is under construction in CIBA aiming for fast nanofabrication at the single-digit nano meter regime [4].

References:

[1]  Ce Zhang, Kai Jiang, Fan Liu, Patrick S. Doyle, Jeroen A. van Kan and Johan R.C. van der Maarel Lab Chip, 13 ( 2013) 2821.

[2] F. Liu, L. Jiang, H. M. Tan, A. Yadav, P. Biswas, J. R. C. van der Maarel, C. A. Nijhuis, J. A. van Kan, Biomicrofluidics 10 (2016) 064105.

[3] JA van Kan, P Malar, and AB de Vera, Rev. Sci. Instrum., 83, (2012) 02B902-1.

[4] X. Xu X, N. Liu N, PS Raman, S. Qureshi, R. Pang, A. Khursheed and JA van Kan, NIMB DOI: 10.1016/j.nimb.2016.12.031.

This paper reports a microfluidic system which can continuously sort motile sperms from semen sample based on sheath flow and the motility of sperms. Besides, we also dilute the normal semen samples to imitate the oligozoospermia patient for the viability enhancement and sorting efficiency by using the developed microfluidic chip.

Recently, cell-based diagnostics and therapeutics are emerging as alternatives for more traditional molecular diagnostics and therapeutics. Various microfluidic tools can be utilized for advancing novel modalities of diagnostics, as well as for enhancing the efficacy and specificity of cell therapy. In this talk, I will showcase some of our recent efforts in this area.

While circulating tumor cells (CTCs) are widely recognized as more specific alternatives for cancer diagnostics, there are many scientific challenges remaining, such as regarding how to define CTCs, or how to extract clinically actionable information from those cells. We have demonstrated the use a microfluidic in vitro culture system for CTCs, as an alternative to simple CTC enumeration, to extract prognostic information and to profile drug dose-response.

In cell therapy, two key engineering bottlenecks are generating sufficient number of cells required, and ensuring the ‘purity’ of product that are truly efficacious. High throughput cell sorting methodologies are ideally suited to address these challenges. I will showcase the examples of such application, in mesenchymal stromal cells (MSCs) and cartilage repair.

Micro- and nanofabricated devices with feature sizes comparable to biological cells are ideal platform for manipulating biological cells for advanced applications. In this talk, we present several microfluidic devices to achieve: 1) deterministic filtering and fractionation of cells according to their sizes for cancer diagnostics; 2) trapping of individual cells of different types for heterotypic cell fusion for biological research; and 3) surface micro and nanostructures for controlling cell growth. In this talk, we focus on the device design, fabrication and testing. Current devices are implemented with traditional channel-based continuous fluid flow. The possibility of achieving similar functionalities using droplet-based active-matrix-driven digital microfluidics will also be discussed.

The synthetic solid-state nanochannels show a broad range of gating and rectification properties that can be further used to implement nanofluidic logic devices similar to the conventional electric logic circuits.¹

Employed the single ion-track-etched nanochannels, which are excellent platforms for studying the ionic transport behavior in nanoscale, we experimentally characterized the nanoscale ionic transport with several different solutions, and found, for the first time, that the ionic conductivity could exhibit an intriguing hysteresis behavior. The hysteresis behavior could be further employed to realize nanofluidic memristors, which are a new type of nanofluidic logic devices² and have been considered as the fourth fundamental elements in conventional electrical circuitry. As nanofluidic memristors, the devices show excellent repeatability, high ON/OFF ratio, and long retention time. Then we further studied the mechanism of the system and required properties of the nanopore. Realizing the memristor concept in nanofluidic regime will not only enrich the family of nanofluidic logic devices, but also greatly enable the nanofluidic circuitry to achieve more complex functionalities.

Early cancer diagnosis, critical to the increase survival rate of cancer patients, is still challenging although to date more than 745,000 relevant published research articles (Google Scholar database) and more than 346,000 articles (PubMed database). In recent years, the detection of Circulating Tumor Cells (CTCs) from patients’ blood samples using microfluidics is a popular non-invasive method for diagnosis and therapy monitoring of cancer. However, the translation from the research of microfluidic CTC devices to a practical CTC detection system in clinics is still challenging because of the significant variation of biological properties among different patients. From the viewpoint of nonlinear dynamic system engineering, we propose a novel methodology: new nonlinear Microfluidic Elasto-Filtration (MEF) model for patients using hyperelasticity for prediction of large deformation of CTC and biophysical measurements of blood samples from each patient for the estimation of equivalent energy dissipation parameter. In this approach, we demonstrate the patient-specific Microfluidic Elasto-Filtration (psMEF) CTC technology can overcome the barrier of translation research. The pre-clinical study of psMEF CTC technology has shown the excellent clinical sensitivity and clinical specificity according to US FDA regulation. Increasing number of clinical data can enable the continuous improvement of psMEF methodology for early cancer diagnosis.

Agriculture of the digital era is going through radical technological changes. Many efforts have been made to make the most of the potential offered by plants and to understand processes happening inside an individual plant. The final goal of our project is to build a “smart field” capable of monitoring the growth process and of controlling the plant growth (Fig.1). To achieve it, we are going to develop an ultra-small nutrients analyzer, a compact 3D-monitor, and an ultra-light environment sensor (light intensity, temperature, humidity, CO2, etc.), which can be installed near plants. Accordingly, plant growth estimation technologies based on micro-fluidic circuit model simulating plant vascular system are being developed. The model will be frequently corrected by using updated data given by those monitors. Furthermore, by making use of the estimation technologies, we try to find out suitable cultivation condition for producing plants having aimed characters.　

This research is supported by JST CREST Grant Number JPMJCR154.

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships.

Dielectrophoresis (DEP) has been extensively used in lab-on-a-chip systems for trapping, separating and manipulating micro particles suspended in liquid medium. In this paper, we report the size-dependent dielectrophoretic crossover frequency of spherical micro polystyrene (PS) particles in Distilled Deionized water (DD-water) and solutions of different conductivity. We apply several methods to obtain the relationship between crossover frequency and medium conductivity. Then, from these results, we provide a better understanding of DEP phenomenon, and explain what factors contribute to the precipitous drop of crossover frequency for increasing medium conductivity.

   Our prior study on size-dependent dielectrophoretic crossover frequency was published in BIOMICROFLUDICS [1], which mainly compared the crossover frequency obtained from dipole model theorem and Maxwell stress tensor (MST), and concluded that, for larger particle size (D > 4.6μm), the dipole model cannot provide accurate prediction. Green [2] and Wei [3], among others, conducted experiments and observed the relationship between crossover frequency and particle size. However, the mechanism that affect the variation of crossover frequency, as a function of particle size and medium conductivity, is not well understood. Here, we conduct systematical study on DEP properties using analytical dipole model, finite element simulations based on MST, and optical tweezers apparatus. From these methods, we obtain an overall understanding of DEP properties from different view, and build a model by PI theorem to predict the result well.

   In Fig. 1, we compare the crossover frequency over medium conductivity obtained from dipole model, MST, and experiment. We observe that, in specified particle size, both MST and experiment have the same trend. From our previous paper, we have known dipole model does not provide accurate prediction for larger particle size, and now we find out, while varying medium conductivity, the result of dipole model also deviate from experiment and simulation. From the simulations using MST, we calculate the surface charge distribution of different medium conductivity (Fig. 2), and notice that, for higher medium conductivity, the surface charge distribution differs much from an ideal dipole, which violates the basic assumption of dipole model theorem. In addition, from experiment result (Fig. 3), we observe that, for higher medium conductivity, the effect of particle size on crossover frequency becomes weaker. We deduce that as medium conductivity increases, charge screening effect becomes dominant [4], which increases the dependence of the crossover frequency on medium conductivity and decrease its dependence on the particle diameter.

In this paper, we present our study on a new type of passive micromixer based on a washboard microfluidic configuration. Periodic geometrical barriers like washboard are built inside a microfluidic channel that alters the flow patterns transversely and vertically. The advantages of this type of mixer is its mixing barriers are at the bottom of the microfluidic channel, and it does not need a complex 2-D or 3-D configurations to perform mixing process. This micromixer can easily be fabricated by one step SU-8 photolithographic process and one molding process. Solutions to be squeezed vertically and laterally while encounter the periodic barriers. Thus, the laminar flow pattern is distorted to create mixing process. To study the mixing mechanism of the skew corrugated micromixer, we study the mixing efficiency of washboard structures at three different angles, including 30, 45, and 60 degrees. Finite element simulations are conducted to study the mixing pattern and efficiency. Simulation results suggested that a skew corrugated microchannel with 45^o angle can provide highest mixing efficiency, and a 95% mixing efficiency can be achieved within 8 stage within a 2.38 mm long microchannel.