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,
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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. 1(b). The design challenges for a femtoliter volumetric pipette include the 11 12 13 need for precise volume control and (2) a surface tension effect in the nanochannel as a replacement 14 15 16 for gravity. With regard to the latter topic, Laplace nanovalves were selected to utilize the so-called 17 18 19 Laplace pressure caused by the liquid surface tension.19 As depicted in Fig. 1(b), the Laplace pressure 20 21 22 exerts a force on the gas/liquid interface, and the Laplace pressure is governed by the surface 23 24 25 wettability and the channel size as represented by the Young-Laplace equation: 26 27 ퟐ 휸 풄풐풔휽 28 푷 = ― #(ퟏ) 29 풓 30 31 where is the surface tension, 휃 is the surface contact angle and r is the equivalent radius of the 32 33 34 channel (푟 = 퐻푊/(퐻 + 푊) where H and W are height and width of nanochannel respectively). When 35 36 37 the surface is hydrophobic (휃 > 90° ), P takes a positive value and works as a pressure barrier against 38 39 40 the introduction of liquid. For operation of the femtoliter volumetric pipette, two different Laplace 41 42 43 valves were configured for use in the nanofluidic device. Laplace valve 1 utilized a difference in 44 45 46 surface wettability and was used to halt the sample solution when no pressure was applied (initial state). 47 48 49 Laplace valve 2 was situated in the main channel to define the sampling volume. To precisely control 50 51 52 the sampling volume at the femtoliter level, the Laplace pressure was controlled via the channel size. 53 54 55 According to Eq. (1), the Laplace pressure P is inversely proportional to the channel size r; 56 57 58 consequently, a narrower channel has a higher Laplace pressure and can function as a valve. Using a 59 60 6 Page 7 of 25 Analyst
1 2 3 4 nanofabrication procedure it is possible to control the width, depth and length of the nanochannel with 5 6 7 a resolution of 10 nm, which is appropriate for femtoliter volume control. A circular shape was adopted 8 9 10 for the femtoliter flask, since this shape can accommodate and smoothly transport the sample via 11 12 13 pneumatic pressure in the nanochannel where the surface tension dominates gravity. 14 15 16 The operation of the femtoliter volumetric pipette and flask is illustrated in Fig. 1(c). All operations 17 18 19 are controlled by application of external pressure. Initially, without application of pressure, the sample 20 21 22 solution is introduced to the main channel by capillary action but is stopped at the intersection point 23 24 25 of the main channel and the air channel by Laplace valve 1. Then, when a pressure P1 (greater than the 26 27 28 Laplace pressure at the intersection point, PL1, but smaller than the Laplace pressure in the narrow 29 30 31 channel, PL2) is applied, the solution moves up to Laplace valve 2. Next, the solution in the air channel 32 33 34 is removed by applying a pressure P2 from the air channel and a pressure P3 from the right side of the 35 36 37 main channel. Here, P2 is greater than PL2 and less than the sum of PL2 and P3. Consequently, only the 38 39 40 solution from the intersection point of the main channel and the air channel to the Laplace valve 2 is 41 42 43 left in place; this volume (the volume measurement) is at the femtoliter level and depends on the 44 45 46 channel size and the distance to the Laplace valve 2. Finally, the solution is transported to the flask by 47 48 49 removing pressure P3 (transport). 50 51 52 53 54 55 3. Experimental 56 57 58 3.1 Design and fabrication of the femtoliter volumetric pipette and flask 59 60 7 Analyst Page 8 of 25
1 2 3 4 As shown in Fig. 2(a), the volumes of the femtoliter volumetric pipette and flask were designed to 5 6 7 be 11 fL and 50 fL, respectively. The femtoliter volumetric pipette had a width of 1600 nm for the 8 9 10 wider channel and 900 nm for the narrower channel. The diameter of the femtoliter flask was 10 m 11 12 13 and the width of the downstream channel was 2100 nm. The depths of the femtoliter volumetric pipette 14 15 16 and flask were 700 nm. In this design, PL1 and PL2 were estimated to be 100 and 130 kPa, respectively, 17 18 19 assuming = 110° (contact angle measured in bulk octadecylsilane at surface) and = 72.75 mN/m 20 21 22 in Eq. (1). 23 24 25 The nanofluidic device was fabricated using a process described elsewhere 8. A Pyrex substrate (0.7 26 27 28 mm thick, 70 mm wide and 30 mm long) with microchannels (500 m wide and 100 m deep) 29 30 31 fabricated by photolithography and wet etching was purchased from the Institute of Microchemical 32 33 34 Technology Co. Ltd., Japan. Nanochannels were fabricated in fused silica (0.17 mm thick, 70 mm 35 36 37 wide and 30 mm long) by electron beam lithography and reactive ion etching. Then, the two substrates 38 39 40 were treated with Piranha solution and oxygen plasma and bonded before being heated at 110°C for 3 41 42 43 hours. The fabricated device is shown in Fig. 2(b). The width and depth of the nanochannels were in 44 45 46 agreement with the target values. 47 48 49 The surfaces of the nanochannels were then modified with hydrophobic molecules 50 51 52 (octadecylsilane). Octadecyldimethyl-N,N-diethylaminosilane (ODS-DEA), synthesized from 53 54 55 diethylamine and octadecyldimethylchlorosilane (ODS-Cl), was used according to a procedure 56 57 58 described elsewhere.20 First, all of the nanochannels were flushed with toluene at 70°C for 30 min. 59 60 8 Page 9 of 25 Analyst
1 2 3 4 Then 20% (v/v) ODS-DEA/toluene was introduced from the bottom side of the vertical channel at 5 5 6 7 kPa and 70°C for 4 hours, flushing the solution once after two hours. After this modification, the 8 9 10 channels were washed with toluene, hexane, acetone, ethanol and water (in that order) and dried by 11 12 13 introducing air to all of the channels. Then, a part of nanochannel was silanolized by introducing 1M 14 15 16 sodium hydroxide. The boundary between the sodium hydroxide solution and the air was controlled 17 18 19 by adjusting the air pressure. Once the boundary reached the intersection point, the pressure was set at 20 21 22 5 kPa for 15 min. After silanolization, all nanochannels were washed with water and dried by passage 23 24 25 of air. 26 27 28 3.2 Experimental setup 29 30 31 A schematic of the experimental setup for the operation of femtoliter volumetric pipette and flask 32 33 34 is given in Fig. 2(a). An inverted microscope (IX71, Olympus, Japan) equipped with a CMOS camera 35 36 37 (ORCA, Hamamatsu Photonics K.K, Japan) was used to observe the movement of solution in the 38 39 40 nanochannels. An LED light source (Lumencor, USA) was used for excitation of sample fluorescence. 41 42 43 The fabricated nanofluidic device was fixed on an aluminum chip holder and placed on the microscope 44 45 46 stage. The inlets of the microchannels were connected with PEEK (polyether ether ketone) tubing via 47 48 49 O-rings and Teflon screws (UF-C, Institute of Microchemical Technology Co. Ltd, Japan). The PEEK 50 51 52 tubing was connected to vials containing reagents, which were linked to pressure controllers 53 54 55 (MFCSTM-EZ, Fluigent, France), and an air compressor. Water, air and surface-modification reagents 56 57 58 were introduced into the microchannels and nanochannels through the PEEK tubing, according to a 59 60 9 Analyst Page 10 of 25
1 2 3 4 programmed pressure-control sequence. The outlets of the microchannels were either connected with 5 6 7 PEEK tubing for discharge to waste or sealed with Teflon screws for precise pressure control. 8 9 10 11 12 13 4. Results and Discussion 14 15 16 4.1 Confirmation of the stop/go function of the Laplace nanovalves 17 18 19 An illustration of the stop/go mode of operation is given in Fig. 3. To check the stop/go function of 20 21 22 the Laplace valves, water was introduced into the device, and the movement of the water/air interface 23 24 25 was observed as the air pressure was increased. When the applied pressure was 70 kPa, the air/water 26 27 28 interface remained at Laplace valve 1, while the interface moved forward when the applied pressure 29 30 31 was over 80 kPa. Thus, the stop/go function of Laplace valve 1 was confirmed. The stop/go function 32 33 34 of the Laplace valve 2 was next investigated. When the applied pressure was adjusted to 140 kPa the 35 36 37 water/air interface was at Laplace valve 2, while the interface moved beyond this point when the 38 39 40 applied pressure attained 150 kPa. Thus, the stop/go function of Laplace valve 2 was confirmed. The 41 42 43 estimated Laplace pressures on Laplace valves 1 and 2 were 70-80 kPa, and 140-150 kPa respectively, 44 45 46 while the respective theoretical values were 100 kPa and 130 kPa. The estimated Laplace pressure on 47 48 49 Laplace valve 1 was 20-30 kPa lower than the theoretical value. This difference in pressure may be 50 51 52 attributed to the mitigation of surface hydrophobicity with the sodium hydroxide treatment. In the case 53 54 55 of Laplace valve 2, the estimated Laplace pressure was 10-20 kPa higher than the theoretical value. 56 57 58 59 60 10 Page 11 of 25 Analyst
1 2 3 4 The surface roughness of the nanochannel (10 nm) may have caused an enhancement of the 5 6 7 hydrophobicity. 8 9 10 4.2 Operating conditions for the femtoliter volumetric pipette 11 12 13 The operating conditions for the femtoliter volumetric pipette were next investigated. After water 14 15 16 was introduced to the femtoliter volumetric pipette, a pressure of 400 kPa was applied to the air channel 17 18 19 to cut and isolate the sample. As shown in Fig. 4(a) top, although the sample was successfully cut, it 20 21 22 disappeared within 4 s of cutting. There are two possible explanations for this phenomenon: leakage 23 24 25 of the water (loss of both solute and solvent) or evaporation (loss of solvent). In an attempt to clarify 26 27 28 the reason for the sample loss, the same operation was conducted but with the use of a fluorescent 29 30 31 solution as the sample. As shown in Fig. 4(a) bottom and 5, the fluorescence intensity of the sample 32 33 34 remained constant, but the total volume of the solution decreased dramatically. Based on the 35 36 37 consideration that the fluorescence intensity corresponded to the number of fluorescence molecules 38 39 40 contained within the solution, it was concluded that evaporation was responsible for the decrease in 41 42 43 solvent while the amount of solute within the nanochannel was retained. Noted that decrease of the 44 45 46 fluorescence intensity after 3 s as shown in Fig. 5 is considered to be because of concentration 47 48 49 quenching by high concentration of fluorescence molecules with the loss of solvent. This evaporation 50 51 52 was attributed to airflow caused by the applied pressure. When 400 kPa was applied to the air channel, 53 54 55 all of the air (nanoliter volume) passed over the sample solution (femtoliter volume) in seconds, 56 57 58 59 60 11 Analyst Page 12 of 25
1 2 3 4 causing evaporation to occur. This suggests that when the sample volume is at the femtoliter level, as 5 6 7 is often the case in nanofluidic devices, evaporation by airflow would be a significant issue. 8 9 10 To overcome the evaporation issue, static pressure was adopted (Fig. 4(b)) rather than dynamic 11 12 13 pressure (Fig. 4(a)). As shown in Fig. 4(b), applying air pressure from both sides of the air channel 14 15 16 minimized the overall air flow (static pressure). The time-course for the sample volume in this 17 18 19 approach is shown in Fig. 6. Compared to the operation using dynamic pressure, the use of a static 20 21 22 pressure effectively reduced the rate of evaporation by ca. 10 times. 23 24 25 Consequently, static pressure was selected as the operating mode for the femtoliter volumetric 26 27 28 pipette. Also, the operating time was set as 0.7 s so that the loss of sample solution by evaporation was 29 30 31 kept to a minimum (less than 1%) as shown in Fig. 6. 32 33 34 4.3 Demonstration of femtoliter volumetric pipette and flask 35 36 37 To demonstrate the working principle of the femtoliter volumetric pipette and flask, the operation 38 39 40 of sampling defined in the section 4.2 was conducted using pure water as a model sample. Fig. 7(a) 41 42 43 shows the time-course microscopic images for femtoliter-scale sampling and transport. First, water 44 45 46 was introduced to the femtoliter volumetric pipette by application of pressure P1 = 100 kPa (Fig. 7(a) 47 48 49 top). Then, air was introduced from both the upper and lower sides of the air channel by application 50 51 52 of a pressure of P2 = 200 kPa, while applying a back pressure of P3 = 140 kPa from the right side of 53 54 55 the main channel (Fig. 7(a) middle). After 0.1s, pressure P3 was turned off and the sample solution 56 57 58 was transported to the femtoliter flask (Fig. 7(a) bottom). As shown in Fig. 7(a), the liquid sample was 59 60 12 Page 13 of 25 Analyst
1 2 3 4 successfully introduced, cut, and transported to the flask. Thus, the working principle of the femtoliter 5 6 7 volumetric pipette and flask was verified. Since the effect of airflow on the evaporation of liquid 8 9 10 sample was minimized, the sampling of 11 fL, which is 85 times smaller than our most recent report,18 11 12 13 was realized. 14 15 16 Performance of the femtoliter volumetric pipette was quantitatively evaluated. Here, the variation 17 18 19 of fabrication, surface modification, and liquid operation were considered as error factors. The volume 20 21 22 of sampling chamber of the femtoliter volumetric pipette was 11.74 ± 0.73 fL (CV 6.2%) with repeated 23 24 25 fabrication, whereas the designed volume was 11.20 fL (width × depth × length = 1600 × 700 × 10000 26 27 28 nm). The variation of hydrophobic-surface modification by octadecylsilane (ODS) was 103.3 ± 1.0° 29 30 31 (CV 1.0%) as the value of the static contact angle. As shown in Fig 7(b), the variation of the measured 32 33 34 sampling volume was 10.83 ± 0.06 fL (CV 0.6 %) based on pixel counting for 10 repeated liquid 35 36 37 operations when the volume of fL sampling chamber was 11.06 fL (width × depth × length = 1580 × 38 39 40 700 × 10000 nm). Effect of above factors on the accuracy and precision of femtoliter volumetric pipette 41 42 43 were discussed. The size variation of fabrication affects the accuracy but not the precision of the 44 45 46 sampling volume because the femtoliter volumetric pipette is integrated in a nanofluidic device and 47 48 49 repeatedly used. The variation of surface modification can affect the Laplace pressure up to 9.8 kPa, 50 51 52 but as the pressure tolerance of Laplace valve 1 and 2 are designed 25.1 kPa, it does not affect the 53 54 55 operational pressure. Thus, in principle, the variation of surface modification does not affect the error 56 57 58 of sampling. In contrast, the variation of liquid operation can affect both accuracy and precision of 59 60 13 Analyst Page 14 of 25
1 2 3 4 sampling volume owing to the different meniscus volume of the air/liquid interface at each time of 5 6 7 operation. Comprehensively, the accuracy of sampling volume was affected by the difference of 8 9 10 fabricated volume (+4.6%) and sampled liquid volume by operation (-2.1%) from the designed volume 11 12 13 while the precision was affected by the variation of sampled liquid volume by operation (0.6%). 14 15 16 Although the precision was three times larger than that for pipetting in a traditional volumetric analysis 17 18 19 (about 0.2%), we consider this performance is acceptable for quantitative applications at femtoliter 20 21 22 level because the error is less than 1% of sample volume. 23 24 25 In addition to pure water as a model sample used in the present study, the concept of the femtoliter 26 27 28 volumetric pipette and flask was applied to real samples of aqueous solution in our recent report, in 29 30 31 which sampling of cell culture medium and protein standard solutions were demonstrated.18 In contrast, 32 33 34 currently, sampling of organic solvents (e.g. methanol, acetonitrile, etc), is difficult because the surface 35 36 37 of the nanochannel is hydrophobic but oleophilic. In the future, sampling of organic solvents can be 38 39 40 achieved by applying oleophobic surface21 to a surface of femtoliter volumetric pipette. On the other 41 42 43 hand, in many cases of analytical chemistry, it is necessary to measure different calibrated volumes. 44 45 46 Regarding a usage of the femtoliter volumetric pipette as a graduated pipette, a multiple of 11 fL (the 47 48 49 sampling volume of current femtoliter volumetric pipette) is feasible by repeating the sampling 50 51 52 operation with desired times. 53 54 55 Utilizing the femtoliter sampling method, high-performance nanofluidic analytical devices 56 57 58 exploiting single-molecule ELISA13 and attoliter-to-femtoliter chromatography with one-million 59 60 14 Page 15 of 25 Analyst
1 2 3 4 theoretical plates11 can be realized. For example, we have realized the quantification of countable- 5 6 7 number proteins secreted from single cell, which is an important achievement for single cell life 8 9 10 sciences.18 Accordingly, the present study developed the femtoliter sampling method in nanochannel, 11 12 13 which has been missing part of three components of analysis (sampling, chemical processing and 14 15 16 detection) in nanofluidics, and will lead to novel analytical applications such as single-molecule or 17 18 19 single-cell analyses. 20 21 22 23 24 25 5. Conclusions 26 27 28 In the context of nanofluidics, we developed a femtoliter volumetric pipette and flask which utilize 29 30 31 air pressure and surface tension for operation. The nanofluidic device incorporating an 11 fL 32 33 34 volumetric pipette and a 50 fL flask was fabricated in glass substrates and reliable operation conditions 35 36 37 required for manipulation of the gas/liquid interface in the nanochannels using Laplace valves was 38 39 40 established. Initially, evaporation of the liquid sample owing to the airflow generated during cutting 41 42 43 of the liquid sample was significant, but we suppressed the evaporation by minimizing the airflow and 44 45 46 shortening the operational time to 0.7 s, less than the time for evaporation. Finally, the working 47 48 49 principle of the femtoliter volumetric pipette and flask with 11 fL sample volume was verified. The 50 51 52 sampling volume was measured as 10.83 ± 0.06 fL (CV 0.6 %) with repeated operations. Generally, 53 54 55 the sampling process is fundamental in analytical chemistry. The present study realized the sampling 56 57 58 at femtoliter volume in nanofluidic device, which is a key technology for analyses of single cell or 59 60 15 Analyst Page 16 of 25
1 2 3 4 analyses at single-molecule level, and hence advance knowledge in various fields including biology 5 6 7 and medicine. 8 9 10 11 12 13 Conflicts of interest 14 15 16 There are no conflicts to declare 17 18 19 20 21 22 Acknowledgements 23 24 25 Fabrication facilities were provided in part by the Academic Consortium for Nano and Micro 26 27 28 Fabrication of four universities (The University of Tokyo, Tokyo Institute of Technology, Keio 29 30 31 University and Waseda University, JAPAN). The authors gratefully acknowledge financial support 32 33 34 from Grant-in-Aid for JSPS Fellows 17J10031 and Core Research for Evolutional Science and 35 36 37 Technology (CREST) of the Japan Science and Technology Agency (JST): JPMJCR14G1. 38 39 40 41 42 43 References 44 45 46 1 A. Manz, H. M. Widmers and N. Graber, Sensors Actuators B Chem., 1990, 1, 244–248. 47 48 49 2 D. R. Reyes, D. Iossifidis, P. A. Auroux and A. Manz, Anal. Chem., 2002, 74, 2623–2636. 50 51 52 3 M. Tokeshi, T. Minagawa, K. Uchiyama, A. Hibara, K. Sato, H. Hisamoto and T. Kitamori, 53 54 55 Anal. Chem., 2002, 74, 1565–1571. 56 57 58 4 T. Ohashi, K. Mawatari, K. Sato, M. Tokeshi and T. Kitamori, Lab Chip, 2009, 9, 991. 59 60 16 Page 17 of 25 Analyst
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Figure 1. Concept of femtoliter-scale sampling method. Conventional volume measurement and 29 30 31 transport in bulk: volumetric pipette and flask (top). femtoliter volume measurement and transport: 32 33 34 femtoliter volumetric pipette and flask (bottom). (b) Operation of femtoliter volumetric pipette and 35 36 37 flask. 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 Analyst Page 20 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Figure 2. (a) Design of femtoliter volumetric pipette and flask. (b) Microscopic and scanning electron 25 26 27 microscopic images of the fabricated femtoliter volumetric pipette and flask. 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20 Page 21 of 25 Analyst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 3. Schematic diagram (top) and microscopic photos (middle and bottom) for investigation of 34 35 36 stop/go function of Laplace valve 1 (a) and 2 (b). 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 21 Analyst Page 22 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 4. Schematic and time-course microscopic images for the operation of femtoliter-scale 26 27 28 sampling with (a) dynamic pressure and (b) static pressure. Rectangle frame (green) represents the 29 30 31 region of interest (ROI) for measurement of fluorescence intensity 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 22 Page 23 of 25 Analyst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 5. Time-course measurement of sample volume and fluorescence intensity. Black circular dots 17 18 19 represent the sample volume in femtoliter volumetric pipette during cutting operation. Green square 20 21 22 dots represent fluorescence intensity from the region of interest (ROI) shown in Fig. 4. 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Figure 6. Time-course measurement of sample volume with the operation using dynamic pressure and 47 48 49 static pressure. 50 51 52 53 54 55 56 57 58 59 60 23 Analyst Page 24 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Figure 7. (a) Demonstration of working principle for femtoliter volumetric pipette and flask. (b) Three 35 36 37 examples of femtoliter liquid sampling (11 fL). The meniscus of the femtoliter liquid sample is the 38 39 40 dominant source of sampling error. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 24 Page 25 of 25 Analyst
1 2 3 4 5 6 7 8 9 10 11 12 13 14 Table of Contents Entry 15 16 17 A femtoliter sampling method, which includes volume measurement and sample transport, was 18 19 20 realized by femtoliter volumetric pipette and flask fabricated on a glass-made nanofluidic device 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 25