Analyst Femtoliter Volumetric Pipette and Flask Utilizing Nanofluidics Journal: Analyst Manuscript ID AN-ART-11-2019-002258.R1 Article Type: Paper Date Submitted by the 26-Dec-2019 Author: Complete List of Authors: Nakao, Tatsuro; The University of Tokyo, School of Engineering Kazoe, Yutaka; The University of Tokyo, Morikawa, Kyojiro; The University of Tokyo, School of Engineering Lin, Ling; National Center for Nanoscience and Technology, Mawatari, Kazuma; The University of Tokyo, Kitamori, Takehiko; The University of Tokyo, Page 1 of 25 Analyst 1 2 3 4 5 Femtoliter Volumetric Pipette and Flask 6 7 8 Utilizing Nanofluidics 9 10 11 1 2 2 1 1,2 12 Tatsuro Nakao , Yutaka Kazoe , Kyojiro Morikawa , Ling Lin , Kazuma Mawatari , and Takehiko 13 14 1,2* 15 Kitamori 16 17 18 1 Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, 19 20 21 Bunkyo, Tokyo 113-8656, Japan 22 23 24 2 Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, 25 26 27 Bunkyo, Tokyo 113-8656, Japan 28 29 30 31 32 33 *Correspondence 34 35 36 Dr. Takehiko Kitamori, Department of Applied Chemistry, The University of Tokyo 37 38 39 6A01 Engineering Building No.3, Hongo 7-3-1, Bunkyo, Tokyo 113-0001, Japan 40 41 42 Email: [email protected] 43 44 45 Phone: +81-3-5841-7231 Fax: +81-3-5841-6039 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 Analyst Page 2 of 25 1 2 3 4 Abstract 5 6 7 Microfluidics has achieved integration of analytical processes in microspaces and realized 8 9 10 miniaturized analyses in the fields such as chemistry and biology. We have proposed a general concept 11 12 13 of integration and extended this concept to the 10-1000 nm space exploring ultimate analytical 14 15 16 performances (e.g. immunoassay of a single-protein molecule). However, a sampling method is still 17 18 19 challenging for nanofluidics despite its importance in analytical chemistry. In this study, we developed 20 21 22 a femtoliter (fL) sampling method for volume measurement and sample transport. Traditionally, 23 24 25 sampling has been performed using a volumetric pipette and flask. In this research, a nanofluidic 26 27 28 device consisting of a femtoliter volumetric pipette and flask was fabricated in glass substrates. Since 29 30 31 gravity, which is exploited in bulk fluidic operations, becomes less dominant than surface effects on 32 33 34 the nanometer scale, fluidic operation of the femtoliter sampling was designed based on utilizing 35 36 37 surface tension and air pressure control. The working principle of an 11 fL volumetric pipette and a 50 38 39 40 fL flask, which were connected by a nanochannel, was verified. It was found that evaporation of the 41 42 43 sample solution by air flow was a significant source of error because of the ultra-small volumes being 44 45 46 processed. Thus, the evaporation issue was solved by suppressing air flow. As a result, the volumetric 47 48 49 measurement error was decreased to ±0.06 fL (CV 0.6%), which is sufficiently low for using in 50 51 52 nanofluidic analytical applications. This study will be a fundamental technology for the development 53 54 55 of novel analytical methods for femtoliter volume samples such as single molecule analyses. 56 57 58 59 60 2 Page 3 of 25 Analyst 1 2 3 4 Introduction 5 6 7 Recently, microfluidics, lab on a chip or micro total analysis systems (-TAS)1,2 have achieved the 8 9 10 miniaturization/ integration of analytical processes into microspaces to realize improved analytical 11 12 13 performance and are expanding in the fields of chemistry and biology. The benefits of integration and 14 15 16 miniaturization include faster reactions due to a higher surface-to-volume ratio, smaller amounts of 17 18 19 reagent and waste, and ease of automation, etc. Our group has proposed a general concept of 20 21 22 integration, called micro-unit operation (MUO).3 In MUO, the chemical process is first divided into 23 24 25 individual operations such as mixing, reaction, extraction, and cell culture, and then each operation is 26 27 28 performed at the micrometer scale. Finally, serial or parallel combinations of MUOs are used to 29 30 31 integrate chemical processes in a microchip system. Nowadays, various kinds of complex chemical 32 33 34 processes including blood tests,4 drug synthesis5,6 and environmental analysis7 have been integrated 35 36 37 into single microchip and microfluidic systems, and such analytical applications are now in a practical 38 39 40 development phase. 41 42 43 The concept of MUO has been further extended to smaller dimensions, i.e., 10-1000 nm on glass 44 45 46 substrates, which we have designated “extended nanospaces”,8 and thus pioneered the field of 47 48 49 nanofluidics. Fundamental technologies for nanofluidics including top-down technology for nano- 50 51 52 fabrication of 10-1000 nm channels on glass substrate9 and highly sensitive methods to detect non- 53 54 55 fluorescent molecules in nanochannels utilizing wave optics and the thermal lens effect were 56 57 58 developed.10 Using these fundamental technologies, we have been seeking the ultimate analytical 59 60 3 Analyst Page 4 of 25 1 2 3 4 method for characterization of single molecules at the femtoliter (fL; 10-15 L) level. For example, a 5 6 7 liquid chromatography (LC) system utilizing pressure-driven flow in a nanochannel was used to 8 9 10 separate a femtoliter sample in a very short time (seconds) with very high efficiency (7,100,000 11 12 13 plates/m), contrasting strongly with that of a commercial high-pressure LC system.11 Also, 14 15 16 implementation of an immunoreaction in an extended nanospace (~fL) permits almost 100% capture 17 18 19 of target proteins utilizing a smaller reaction field than in molecular diffusion (~10 m on a seconds 20 21 22 time scale).12 This efficient reaction field permitted analysis of a single protein molecule by integration 23 24 25 of an enzyme linked-immunosorbent assay (ELISA) into a nanochannel.13 It is anticipated that such 26 27 28 nanofluidics regimes will be eminently suited to novel analytical devices that process ultra-small 29 30 31 sample volumes (e.g., single cell/single molecule analysis). 32 33 34 However, while the analytical process consists of three components, i.e., sampling, chemical 35 36 37 processing and detection, the means for the initial sampling is still challenging in nanofluidic devices 38 39 40 despite its importance. Several researchers have reported downsizing of the sampling method using 41 42 43 microfluidics, but these methods are difficult to implement in nanofluidics. In one case, for example, 44 45 46 aqueous picoliter droplets in an oil phase were generated and manipulated by surface acoustic waves 47 48 49 14,15 or by electrowetting.16 However, use of an aqueous/oil interface can result in cross contamination 50 51 52 whereas application of an electric current can bias sampling. In the work of Huang et.al., a nanoliter 53 54 55 liquid sampling method based on a microfluidic pneumatic valve was proposed,17 which involved 56 57 58 deformation of a soft material (e.g., polydimethylsiloxane) by pneumatic pressure; however, 59 60 4 Page 5 of 25 Analyst 1 2 3 4 fabrication of nanochannels in soft material is problematic using current technology. 5 6 7 In the present study, a sampling method for femtoliter volume measurement and sample transport 8 9 10 was developed. In analytical chemistry, conventional sampling and transfer operations for liquids are 11 12 13 performed at a microliter to liter scale, normally using volumetric pipettes and flasks. In this work, 14 15 16 downscaling of the volumetric pipette and flask to the femtoliter was achieved. Given that gravitational 17 18 19 effects, which are exploited in bulk fluidic operations, become much smaller than surface effects in a 20 21 22 nanospace, we developed fluidic operations of the femtoliter volumetric pipette and flask which 23 24 25 exploited the surface tension of the sample solution. To realize unbiased femtoliter sampling, the 26 27 28 formation of a gas/liquid interface that was free from cross-contamination and controlled by surface 29 30 31 tension and air pressure was required. We note that the concept of femtoliter sampling is incorporated 32 33 34 in our most recent paper to achieve living single-cell protein analysis by nanofluidics.18 Here, we report 35 36 37 device design, principle verification, evaluation of performance, and development of fluidic operation 38 39 40 with avoiding femtoliter sample evaporation. A nanofluidic device, which incorporated an 11 fL 41 42 43 volumetric pipette and a 50 fL volumetric flask, was designed and fabricated, and the performance for 44 45 46 volume measurement and transport was evaluated. 47 48 49 50 51 52 Working principle 53 54 55 Fig. 1 shows schematic diagrams of traditional and femtoliter volumetric pipettes and flasks. In a 56 57 58 conventional volumetric pipette (Fig. 1(a)), a sample solution is sucked up by a mechanical pump until 59 60 5 Analyst Page 6 of 25 1 2 3 4 it reaches the marked calibration line (volume measurement), and is then transported to the flask by 5 6 7 gravity transport. Based on this approach, we propose a femtoliter volumetric pipette and flask that 8 9 10 operates as shown in Fig.