Featuring work from Prof. Yan Xu’s NanoBio Interfaces
Laboratory (NaBIL) at Osaka Prefecture University, which has
been involved in developing nanobio interfaces in nanofluidic
devices for single molecule and single cell studies.

As featured in:

Title: Regeneration of glass nanofluidic chips through a multiplestep sequential thermochemical decomposition process at hightemperatures
A simple method enabling the revival of ‘dead’ glass nanofluidic
chips was described. The method provides an efficient solution
to significantly improve the reusability of nanofluidic chips andwill be useful for the promotion and acceleration of research andapplications of nanofluidics.
See Yan Xu et al. Lab Chip, 2015, 15,3856.

www.rsc.org/loc
Registered charity number: 207890

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Regeneration of glass nanofluidic chips through amultiple-step sequential thermochemical
decomposition process at high temperatures†
Yan Xu,*a Qian Wu,ab Yuji Shimataniab and Koji Yamaguchiac
Due to the lack of regeneration methods, the reusability of nanofluidic chips is a significant technicalchallenge impeding the efficient and economic promotion of both fundamental research and practicalapplications on nanofluidics. Herein, a simple method for the total regeneration of glass nanofluidic chips
was described. The method consists of sequential thermal treatment with six well-designed steps, whichcorrespond to four sequential thermal and thermochemical decomposition processes, namely, dehydration, high-temperature redox chemical reaction, high-temperature gasification, and cooling. The method

Received 31st May 2015,
Accepted 4th August 2015
DOI: 10.1039/c5lc00604j

enabled the total regeneration of typical ‘dead’ glass nanofluidic chips by eliminating physically clogged
nanoparticles in the nanochannels, removing chemically reacted organic matter on the glass surface andregenerating permanent functional surfaces of dissimilar materials localized in the nanochannels. The
method provides a technical solution to significantly improve the reusability of glass nanofluidic chips andwww.rsc.org/loc

will be useful for the promotion and acceleration of research and applications on nanofluidics.

Over the past two decades, microfluidic and nanofluidic
based lab-on-a-chip (LOC) systems have been broadly used forresearch in multidisciplinary fields including chemistry,
biology, medicine and physics.1 LOC systems havedemonstrated their capabilities and powerfulness in a variety
of applications such as microseparation, chemical/biosensing,biohazard detection, environmental monitoring and portableor point-of-care diagnostics, as well as large-scale omics
research, single cell analysis and single molecule studies.2–5
Much progress in terms of device fabrication, component
integration, process control, detection principles, transportmechanisms, and fluidic theories has been achieved. However, due to the lack of regeneration methods, the reusabilityof LOC devices is still a significant practical challenge, whichis common to almost all LOC devices. The challenge not only
impedes the commercialization and widespread use ofaNanoscience and Nanotechnology Research Center, Research Organization forthe 21st Century, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai,
Osaka 599-8570, Japan. E-mail: y-xu@21c.osakafu-u.ac.jpbDepartment of Chemistry, Graduate School of Science, Osaka Prefecture
University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japanc
Department of Quantum and Radiation Engineering, Graduate School ofEngineering, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka
599-8531, Japan† Electronic supplementary information (ESI) available: XPS spectra of sulfur (S2p)
on 3-mercaptopropyltrimethoxysilane (MPTS) modified glass substrates before
and after treatment by the regeneration process (Fig. S1). See DOI: 10.1039/
c5lc00604j

3856 | Lab Chip, 2015, 15, 3856–3861

thriving microfluidics but also affects the efficient promotionof fundamental research on nascent nanofluidics.
Channel wall fouling by undesired adsorption and channel clogging by dust or particles are major problems
preventing the reuse of LOC devices.6 In addition, many LOC
devices enable only single use, especially those devices comprising permanent functional surfaces or units which areused to irreversibly react or interact with target molecules.
These problems more frequently occur and are significantly
severer as the channel sizes further decrease from themicrometer scale to the nanometer scale. For microfluidic
devices, anti-fouling coating,7,8 rinsing at high fluid velocity9or using harsh reagents, and surface regeneration by desorption of molecules via physical,10 chemical,11,12 electrochemical,13 or mechanical14 approaches allow partial alleviation of these problems. However, these methods
unfortunately exhibit limited effects on the total regenerationof the devices because additional reagents are usuallyrequired in most cases but may be difficult to totally remove.
Especially, application of these methods to nanofluidic
devices is not easy, due to various technical hurdles resulting
from the scaling down of the channel structures, where even
operation of liquid introduction becomes significantly more
complicated than that in microfluidic devices.
Nanofluidic devices are promising tools to elucidate chemical, physical, and biological phenomena and processes confined in nanometer-scale spaces at the single-molecularlevel.4,15,16 Fused-silica glass is currently almost the sole ideal
substrate material for the fabrication of nanofluidic devices,

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due to its well-established nanofabrication technologies andsuperior optical, mechanical, thermal, and chemical properties. Besides the high cost of the substrate (e.g., 30–40US$ for
40 mm × 30 mm × 0.7 mm fused-silica glass), glass nanofluidic chips are fabricated using expensive nanofabrication
technologies such as electron beam (EB) lithography integrated with plasma dry etching17,18 or focused ion beamlithography19,20 through delicate, sophisticated and timeconsuming processes performed in clean rooms, and hence
are not affordable for only single use. Therefore, regeneration
methods allowing the reuse of glass nanofluidic chips are
greatly desired to efficiently and economically promote andaccelerate the research and applications on nanofluidics.
Herein, we describe a general methodology for the regeneration of glass nanofluidic chips using a multiple-step thermochemical decomposition process at high temperatures(Fig. 1). The thermochemical decomposition occurs duringwell-designed sequential thermal treatment at controlledtemperatures and pressures in a vacuum furnace (Fig. 1a).
The method takes advantage of the exceptional properties of
fused-silica glass in terms of strong heat resistance (strain
point: 1070 °C; annealing point: about 1140 °C; softening
point: about 1665 °C) and extremely low thermal expansion(coefficient of thermal expansion near zero). These exceptional properties allow numerous thermal treatments of glass
nanofluidic chips at high temperatures (for example, at1000 °C) without changing the nanochannel structures.
Therefore, the sequential thermal treatment at high temperatures enables the revival of ‘dead’ nanofluidic chips by

Fig. 1 Schematic drawing of (a) regeneration of glass nanofluidic
chips through (b) sequential thermochemical decomposition.

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Technical Innovation

totally removing adsorbed dust, clogged particles, andreacted/interacted organic matter in the nanochannels via
thermochemical decomposition. Furthermore, the method
not only allows the total regeneration of glass nanofluidic
chips, but is also appropriate for the regeneration of nanofluidic chips with local functional units and surfaces composed of dissimilar materials such as metallic nanoarrays by
performing the thermal treatment at suitable high temperatures. In addition, the method is simple, as no complicatedoperations are required because all steps of the thermal
treatment can be carried out using a commercial automatic
program-controlled vacuum furnace.
In brief, the method comprises the following six sequential steps of thermal treatment (Fig. 1b): (i) raise the temperature from room temperature (~25 °C) to 100 °C and simultaneously exhaust the air (or waste gases) in the furnacechamber to a level of 101 Pa in 10 min; (ii) maintain the temperature at 100 °C and the vacuum (~101 Pa) for 2 h; (iii) leakthe furnace chamber to atmospheric pressure (~105 Pa) andraise the temperature to a high temperature (600 °C or 1000
°C in this study) in 3 h; (iv) maintain the high temperature(600 °C or 1000 °C) as well as the leak conditions (~105 Pa)
for 6 h; (v) exhaust the air (or waste gases) in the furnacechamber to the level of 101 Pa again and maintain the hightemperature (600 °C or 1000 °C) for 6 h; (vi) maintain thevacuum conditions (~101 Pa) and gradually lower the temperature to room temperature (~25 °C) at the same time by using
the cooling fan of the furnace (>3 h).
These six steps correspond to four sequential thermal andthermochemical processes (Fig. 1b), namely, dehydration(i.e., drying; steps i to ii), high-temperature redox chemical
reaction (i.e., burning; steps iii to iv), high-temperature gasification (step v), and cooling (step vi). First, the dehydration or drying process (steps i to ii) occurring at 100 °C isused to remove the liquids which remained in the channelsof the ‘dead’ nanofluidic chips. Because the resulting
steam may be involved in the subsequent reactions at hightemperatures and consequently bring risks of generation ofsubstances difficult to remove via hydrolysis,21,22 the steam
is always exhausted by a vacuum pump during the first process. Then, the ‘dead’ nanofluidic chips undergo the redox
chemical reaction process under air atmosphere at elevatedtemperatures (steps iii to iv), where adsorbed dust, clogged
particles, and reacted or interacted organic matter in the‘dead’ nanochannels react with atmospheric oxygen,thereby mostly decomposing into gaseous products such ascarbon dioxide, water vapor, hydrogen, and carbon monoxide.23 During this process, a trace amount of ash (carbon orcarbonaceous materials) may be produced, due to incomplete burning. After this, the gaseous products are exhausted
by the vacuum pump, and the trace amount of carbonaceousash can be further converted into exhaustible gaseous products, during the high-temperature gasification process (step v).
Here, gasification is a term usually used in the field of energy
generation and conversion, and in this study, specificallydescribes the thermochemical decomposition of carbonaceous

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Technical Innovation

matter into carbon monoxide, hydrogen, and carbon dioxide
at high temperatures, without burning, in the absence of oxygen.24 Finally, after naturally cooling down to room temperature (step vi), the regenerated nanofluidic chips are ready forreuse.
The multiple-step sequential thermochemical decomposition process enabled the total regeneration of a glass nanofluidic chip with nanochannels heavily clogged by nanoparticles (Fig. 2), which is a typical and frequent caseseriously preventing the reuse of nanofluidic chips when handling nanoparticles in nanochannels. The nanofluidic chip(Fig. 2a) was fabricated on fused-silica substrates (40 mm ×
30 mm × 0.7 mm; Sendai Quartz, Sendai, Japan) according toprocesses reported previously.18,25 The nanofluidic chip comprises 50 parallel-arrayed nanochannels (300 nm deep, 800
nm wide and 400 μm long, spaced by 3 μm) which areconnected to two side microchannels (3 μm deep and 500
μm wide) (Fig. 2b). The chip structure is a standard one usedin nanofluidics to facilitate liquid introduction into the nanochannels through the microchannels.25–27 The entrances andnearby areas on the left sides of the nanochannels (Fig. 2c)
were heavily clogged by 50 nm fluorescent nanoparticles(micromere®-greenF, polydispersity index <0.2; Micromod,
Rostock, Germany), when the nanoparticles at a high concentration of 3.0 × 1011 mL−1 were introduced through the left
microchannel to the central arrayed nanochannels(Fig. 2d and e). The nanoparticles are made of polystyrene
and polymethacrylate and immobilized with fluorescein isothiocyanate (FITC; excitation/emission = 494 nm/518 nm).
Strong fluorescence revealing the clogged areas in theentrances and the nanochannels (Fig. 2e) was observed byusing a fluorescence microscope (BX53, Olympus, Tokyo,
Japan) with an electron multiplying charge coupled device(EM-CCD) camera (iXon3, Andor, Belfast, UK). The chip was

Lab on a Chipthen treated by the multiple-step sequential thermochemical
decomposition process using an automatic programcontrolled vacuum furnace (KDF75, Denken-High Dental,
Kyoto, Japan). Here, we performed steps iv and v of the thermal treatment (Fig. 1b) at 1000 °C, considering that the chip
is a totally glass chip without other functional units not suitable for high-temperature treatment. As shown in Fig. 2f, nofluorescence was detected on the previously clogged areas
after treatment, suggesting that the polymer nanoparticles
might be totally removed from the channels by hightemperature thermochemical decomposition. To further confirm this, the introduction of a solution (50 μM) of a fluorescent dye (acid red 52, excitation/emission = 565 nm/585 nm;
TCI, Tokyo, Japan) from the left microchannel to the central
arrayed nanochannels by air pressure was performed using acustom-made pressure-driven nanofluidic control system.18,25
The solution could be smoothly introduced from the left
microchannel through the central arrayed nanochannels to
the right microchannel, even at a low operation pressure of
50 kPa. Homogeneous fluorescence in all nanochannels wasobserved during the introduction (Fig. 2g), revealing that
there were no clogged nanochannels in the chip. Furthermore, the channel electrical conductance of another glass
nanofluidic chip with a similar structure (100 parallel-arrayed
nanochannels) was characterized before and after regeneration using the experimental setup shown in Fig. 2h. The electrical conductance was obtained by measuring the electricalresistance, which is the inverse quantity of the electrical conductance. The result (Fig. 2i) shows that the electrical conductance of the channels (fully filled with 0.1 M KCl solution,measured at 25 °C), after being heavily clogged by the nanoparticles (3.0 × 1011 mL−1, in 0.1 M KCl solution), decreased
significantly, but after regeneration, the electrical conductance recovered to the same level as that of the new chip.

Fig. 2 Evaluation of the regeneration of the glass nanofluidic chip with nanochannels clogged by fluorescent nanoparticles (50 nm). (a) A pictureof the glass nanofluidic chip; (b, c) schematic drawings of the structure of the nanofluidic chip and the observation area for (d–g); (d–e) a brightfield image and the corresponding fluorescence image of the observation area indicating nanochannel clogging by the fluorescent nanoparticles;(f) a fluorescence image of the observation area after the regeneration; (g) a fluorescence image and a partial enlarged view (with brightness/contrast adjusted for better visibility) of the regenerated channels during introduction of acid red 52 (50 μM). (h) The experimental setup for the measurement of (i) the channel electrical conductance at 0.1 M KCl for the new chip, the clogged chip, and the regenerated chip.

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These results indicate that clogging in the nanofluidic chipby the polymer nanoparticles was totally removed using thechip regeneration method. We still use the chip which has
been regenerated at least ten times using the same regeneration process. Therefore, we consider that the regeneration
method allows numerous times of reuse of totally glass nanofluidic chips with physical clogging by typical organic matter.
In addition, the method also exhibited the capability toremove organic matter chemically reacted on glass surfaces
via salinization, which is a versatile chemical surface modification method to fully or partially modify the walls of glass
micro/nanochannels.8,28,29 For example, X-ray photoelectron
spectroscopy (XPS; ESCA-3200, Shimadzu, Kyoto, Japan) analysis of 3-mercaptopropyltrimethoxysilane (MPTS; Nacalai
Tesque, Kyoto, Japan) silanized glass substrates revealed that
the MPTS layers were removed after treatment by the regeneration process (steps iv and v at 1000 °C) because the sulfur
2p peak (S2p) specifically attributed to MPTS disappeared
after regeneration (Fig. S1 in the ESI†).
The revival of ‘dead’ nanofluidic chips containing local
permanent functional surfaces of dissimilar materials was
also achieved by using the chip regeneration method (Fig. 3).
The nanofluidic chip (Fig. 3a) used in this experiment alsopresents a standard micro/nanochannel hybrid structure, buteach nanochannel (300 nm deep, 800 nm wide and 400 μm

Technical Innovation

long, spaced by 2 μm) contains pre-deposited gold nanoarrays (500 nm square, spaced by 2 μm; thickness of Au/Cr =45 nm/5 nm) (Fig. 3b). The chip was fabricated using amethod for site-specific nanopatterning in nanofluidic channels which we developed.30 The chip was used to form molecular nanoarrays in arrayed nanochannels by bottom-up
molecular self-assembly based on notable thiol–gold or disulfide–gold interactions. On the gold nanoarrays in the arrayed
nanochannels (Fig. 3c), fluorescent self-assembled monolayer(SAM) nanoarrays (Fig. 3d) were first formed using a fluorescein disulfide, HOOC–ĲCH2)10–S–S–ĲCH2)10–CONH–fluorescein(ProChimia Surfaces, Gdansk, Poland), according to a process
which we previously described.30 Fluorescein disulfide isfamous for the formation of stable fluorescent SAMs on gold
via the semi-covalent sulfur–gold bond, with strong bondstrength in the order of 45 kcal mol−1.31 Once the fluorescent
SAMs form, they are difficult to remove. For example, theformed fluorescent SAM nanoarrays in the arrayed nanochannels were still detectable using a fluorescence microscope (BX53, Olympus, Tokyo, Japan), even after being
subjected to continuous strong fluidic rinsing by flowing
absolute ethanol under high pressure at 430 kPa for six days.
Hence, without the regeneration of gold array surfaces forreforming the SAMs, the chip was hardly used again after the
single assay experiment. The chip with the fluorescent SAM

Fig. 3 Evaluation of the regeneration of the glass nanofluidic chip with gold nanoarrays in arrayed nanochannels. (a) A picture of the nanofluidic
chip; (b) schematic drawing of the cycle of SAM formation, chip regeneration, and SAM reformation; (c) bright-field and (d) fluorescence images ofthe fluorescent SAM nanoarrays formed on the gold nanoarrays before the regeneration, (e) bright-field and (f) fluorescence images of the gold
nanoarrays after the first regeneration; (g) a fluorescence image of the reformed fluorescent SAM nanoarrays after the first regeneration; (h)bright-field and (i) fluorescence images of the gold nanoarrays after the third regeneration; (j) a fluorescence image of the reformed fluorescent
SAM nanoarrays after the third regeneration; (k) XPS spectra of sulfur (S2p) on the gold-deposited glass substrates with the fluorescent SAMs before
and after treatment by the same regeneration process.

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nanoarrays (Fig. 3d) was treated by the regeneration method(Fig. 3b), where steps iv and v of the thermal treatment(Fig. 1b) were performed at 600 °C. The temperature wasdeliberately set to be much below the melting point of gold
(1064 °C) so that the organic SAMs on the gold nanoarrays
could be thermochemically decomposed while the gold
nanoarrays were still retained. Fig. 3e and f show a brightfield image and a fluorescence image of the nanoarrays in
the arrayed nanochannels after regeneration for the firsttime, respectively. While the gold nanoarrays were clearlyobserved under the bright field (Fig. 3e), no fluorescence
was detected on the gold surfaces, implying that the fluorescent SAMs might be removed by the regeneration. The regeneration was further confirmed at the elemental level by XPS
analysis. Because it is difficult to directly examine the surfaces in the closed small nanochannels by using XPS, in thisexperiment, the fluorescent SAMs formed on the golddeposited glass substrates (5 mm × 5 mm × 0.7 mm; Sendai
Quartz) under the same conditions as those in the nanochannels were examined before and after treatment. As
shown in Fig. 3k, an XPS signal at ~164 eV specifically attributed to the sulfur 2p peak (S2p) was detected before theregeneration treatment but not after the regeneration treatment. The result indicates that the fluorescent SAMs weretotally removed from the gold surfaces via cleavage of thesulfur–gold bond by the treatment. A similar result was
obtained in another XPS determination on hydrophobic
1-butanethiol (BT; Nacalai Tesque) SAMs formed on the golddeposited glass substrates before and after the regeneration
treatment (Fig. S2a in the ESI†). Accordingly, the BT/Au
hydrophobic surfaces (contact angle = 73.9°) recovered tohydrophilic surfaces (contact angle = 29.1°), which was at
the same level of bare gold surfaces (contact angle = 28.3°)
after being subjected to the same regeneration process (Fig.
S2b in the ESI†). These results reveal that the treatment
enabled the regeneration of the gold surfaces in the nanochannels by totally removing the organic matter which specifically interacted on the gold surfaces. Furthermore, thefluorescent SAM nanoarrays could be formed again on theregenerated gold nanoarrays in the arrayed nanochannels(Fig. 3g). The cycle of SAM formation, chip regeneration, andSAM reformation (Fig. 3b) was performed three times andthe total regeneration and SAM reformation were confirmed
in each cycle. While Fig. 3h and i show a bright-field image
and the corresponding fluorescence image (no fluorescence
was detected) of the gold nanoarrays after regeneration for
the third time, respectively, Fig. 3j demonstrates a fluorescence image of the fluorescent nanoarrays successfullyreformed after the third regeneration. It should be notedthat the gold nanoarrays maintained their shining goldencolor after regeneration for the second time (Fig. S3†), but
exhibited a dark red color after the third regeneration(Fig. 3h), when being observed under the same bright-fieldmicroscopic conditions. Such change probably resulted fromthe well-known phenomenon in which the chromium atoms
diffuse to the gold surface during thermal treatments.32

3860 | Lab Chip, 2015, 15, 3856–3861

Lab on a Chip

In conclusion, the use of a well-designed sequential thermochemical decomposition process comprising dehydration,
high-temperature redox chemical reaction, high-temperature
gasification, and cooling steps to regenerate glass nanofluidic
chips was described. The capabilities of the method to totally
remove physically clogged nanoparticles as well as chemicallyreacted organic matter and effectively regenerate local permanent functional surfaces of dissimilar materials in the nanochannels were evaluated and confirmed both from the aspectof chip functions and at the elemental level. Accordingly, thetotal regeneration of ‘dead’ glass nanofluidic chips withthese typical problems which usually prevent the reuse ofglass nanofluidic chips was achieved. The use of the method
can significantly improve the reusability of glass nanofluidic
chips, which has been widely desired but greatly challenging.
Therefore, we believe that this method will be very useful forthe efficient and economic promotion of both fundamental
research and practical applications on nascent nanofluidics.

Acknowledgements
This work was partially supported by JSPS KAKENHI (grant
numbers 26706010 and 26630403), MEXT KAKENHI (grant
number 26107714), and the Asahi Glass Foundation. The
authors thank Prof. Hiroshi Inoue and Prof. Eiji Higuchi at
Osaka Prefecture University for their assistance in XPS
determination.

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