\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Ferrowax microvalve-utilized centrifugal microfluidic system for a fully
automated serial dilution}



\author[1]{Soohyun Kim}%
\author[1]{Jayeon Song}%
\author[1]{RaKyeom Kim}%
\author[2]{Nae Yoon Lee}%
\author[3]{Min Hwan Kim}%
\author[4]{Hyun Gyu Park}%
\affil[1]{Korea Advanced Institute of Science and Technology}%
\affil[2]{Gachon University}%
\affil[3]{Biomedicus}%
\affil[4]{Korea Advanced Institute of Science and Technology (KAIST)}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
We herein describe an ingenious centrifugal microfluidic system to
accomplish a fully automated serial dilution. The liquid flow on the
disc was automatically regulated by utilizing ferrowax microvalves
systematically integrated into the channels within the specially
designed metering structure. By opening the differently positioned
microvalves through irradiation of IR laser to allow metering, the same
amount of diluent was serially eluted to the dilution chamber from the
same diluent chamber. After dilution, the diluted samples were
automatically delivered to the respective final product chambers by
appropriately opening or closing the microvalves in the connecting
channels, followed by rotating the disc. Based on this unique design
principle, six consecutive two-fold and ten-fold dilutions were
successfully achieved, yielding excellent accuracy in a wide dynamic
range up to six orders of magnitude. Very importantly, the overall
serial dilution process, including the diluent addition, mixing, and
product transfer steps, was completed very rapidly within five minutes,
due to the minimized procedures enabled by the automated actuation of
the ferrowax microvalves at the rationally designed positions. The
centrifugal microfluidic system would serve as a powerful elemental tool
to realize the fully automated diagnostic microsystem involving the
serial dilution process.%
\end{abstract}%



\sloppy


\textbf{Ferrowax microvalve-utilized centrifugal microfluidic system for
a fully automated serial dilution}

Soohyun Kim\textsuperscript{a,+}, Jayeon Song\textsuperscript{a,+},
RaKyeom Kim\textsuperscript{a,b}, Nae Yoon Lee\textsuperscript{b}, Min
Hwan Kim\textsuperscript{c,*}, and Hyun Gyu Park\textsuperscript{a,*}

\textsuperscript{a} Department of Chemical and Biomolecular Engineering
(BK21+ Program), Korea Advanced Institute of Science and Technology,
291, Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

\textsuperscript{b} Department of BioNano Technology, Gachon University,
1342, Seongnam-daero, Sujeong-gu, Seongnam-si, Gyeonggi-do, 13120,
Republic of Korea

\textsuperscript{c} Biomedicus, 212, Gasan digital 1-ro, Geumcheon-gu,
Seoul, 08502, Republic of Korea

\textsuperscript{+} These authors equally contributed to this work

\textsuperscript{*} Corresponding authors.

E-mail: hgpark@kaist.ac.kr (H. G. Park); Phone: +82-42-350-3932; Fax:
+82-42-350-3910

E-mail: mhkim@biomedicus.co.kr (M. H. Kim); Phone: +82-2-6713-8673

\textbf{Abstract}

We herein describe an ingenious centrifugal microfluidic system to
accomplish a fully automated serial dilution. The liquid flow on the
disc was automatically regulated by utilizing ferrowax microvalves
systematically integrated into the channels within the specially
designed metering structure. By opening the differently positioned
microvalves through irradiation of IR laser to allow metering, the same
amount of diluent was serially eluted to the dilution chamber from the
same diluent chamber. After dilution, the diluted samples were
automatically delivered to the respective final product chambers by
appropriately opening or closing the microvalves in the connecting
channels, followed by rotating the disc. Based on this unique design
principle, six consecutive two-fold and ten-fold dilutions were
successfully achieved, yielding excellent accuracy in a wide dynamic
range up to six orders of magnitude. Very importantly, the overall
serial dilution process, including the diluent addition, mixing, and
product transfer steps, was completed very rapidly within five minutes,
due to the minimized procedures enabled by the automated actuation of
the ferrowax microvalves at the rationally designed positions. The
centrifugal microfluidic system would serve as a powerful elemental tool
to realize the fully automated diagnostic microsystem involving the
serial dilution process.



\textbf{1. Introduction}

Serial dilution is one of the most essential unit operations in numerous
biological and chemical experiments, which currently normally involves
tedious and time-consuming manual pipetting. The serial dilution by the
labor-intensive manual pipetting, however, is quite influenced by the
user's expertise and the accurate pipetting is not ensured, particularly
when the sample volume is very small down to microliter scale.
Therefore, there is a great incentive existing for the development of an
automated serial dilution technique to minimize the manual efforts and
eliminate the user influence.

Currently, two strategies are being mainly exploited to achieve
automated serial dilutions, which basically rely on automated dispensing
using pipetting machines {[}1-5{]} and microfluidic systems {[}6-12{]}.
The pipetting machine can eliminate user influence and easily control
the number, factor, and volume of dilutions. However, it is relatively
expensive and many pipetting errors are still inevitable as in the
conventional manual pipetting, particularly when handling highly viscous
samples such as general biological specimens or PCR master mixes.

As an alternative approach, the microfluidic systems have been
extensively exploited to achieve an automatic serial dilution by
employing various elaborately designed microchannels and metering
structures. A major advantage of the microfluidic systems is the ability
to precisely manipulate small volume samples, thus reducing reagent
and/or sample loss. The microfluidic systems, however, normally require
device integration with the macroscale environment and essentially
involve external pumps and connecting tubes between the channels and
pumps for fluid control, which would make the system expensive and
complicated. Furthermore, the control of the sample volume or dilution
ratio in the microfluidic systems is quite intricated due to the
continuous propensity of the fluidic flow within the indiscrete
structure {[}9-12{]}. In this regard, the centrifugal microfluidic
system could greatly simplify the microfluidic process by manipulating
liquid flow by rotating the disc at various speeds and in different
spinning directions, and eliminating the requirements for the pumps,
tubings, and associated connectors {[}13-20{]}.

Based on this background, centrifugal microfluidic devices have been
intensively developed to achieve automatic serial dilutions. Most
representatively, \emph{Kim et al.} {[}21{]} developed a centrifugal
microfluidic device capable of automatically conducting arbitrary serial
dilution by utilizing reversible diaphragm valves and specially designed
two metering zones. They demonstrated that the device was able to
accomplish wide dynamic range serial dilutions with excellent accuracy
(R\textsuperscript{2} \textgreater{} 0.97). Juelg \emph{et al.} {[}22{]}
also reported the centrifugal microfluidic system for automated serial
dilutions by utilizing fill-level-coupled valving as a key element. They
successfully accomplished the automated serial dilutions required for
the on-disc construction of qPCR standard curve, verifying the
capability of the system for the serial dilution in a wide dynamic range
with high accuracy and precision.

In this paper, we present another advanced centrifugal microfluidic
system equipped with individually addressable laser-irradiated ferrowax
microvalves (LIFM) {[}23{]}, which enables fully automated serial
dilutions in an accurate, ultrafast, and loss-free manner. To this end,
the capability of the system was successfully verified by accomplishing
six consecutive two-fold and ten-fold dilutions with excellent accuracy.

\textbf{2. Material and methods}

2.1. Design and fabrication of the centrifugal microfluidic disc

The serial dilution disc was designed using computer-aided design
software (AutoCAD, Autodesk, Inc., Sausalito, CA, USA). The disc was
fabricated by assembling three separate layers: a 5 mm polycarbonate
bottom plate (IDEL Corp., Chilgok, Korea), a 1 mm polycarbonate top
plate (IDEL Corp., Chilgok, Korea), and a 0.1 mm middle layer of
double-sided polyimide tape (930H, Coretech, Gimpo, Korea). Figure 1(a)
shows an expanded view of this assembly, while its assembled form is
shown in Figure 1(b). The top and bottom layers were milled by a
computer numerical control machining center (HI-M1300, Hwacheon, Seoul,
Korea). The bottom layer was further treated with a diluted chloroform
solution to obtain a smooth and transparent milled-surface, and both the
top and bottom layers were cleaned with 70\% ethanol and distilled water
(DW). The layers were additionally autoclaved when they were used to
dilute the \emph{Escherichia Coli} O157:H7
(Shigatoxigenic\emph{Escherichia coli} (STEC)) samples. The bottom layer
was mainly composed of six identical sections containing two diluent
chambers, one dilution chamber, seven product chambers, and associated
deep channels, while the top layer was designed to consist of the
appropriately positioned ferrowax grooves, venting holes, inlet holes,
and outlet holes. A double-sided polyimide tape for the middle layer was
fabricated using a cutting plotter (Silhouette Cameo 3, Silhouette
America\selectlanguage{ngerman}\textsuperscript{®} Inc., Orem, UT, USA). The middle layer was
used to bind the top and bottom layers after the areas corresponding to
the chambers and channels are peel off.

To facilitate the convenient and accurate assembly of the three layers,
we employed 3D printed jig fabricated by 3D printing (3DWOX 2X, Sindoh
Co., Ltd., Seoul, Korea) of a polylactic acid filament (Figure S1),
which enabled the accurate alignment of the three layers having a
perforated structure of the central axis and six alignment holes. Prior
to the assembly, the ferrowax prepared by mixing the same volumes of
paraffin wax (T\textsubscript{m} = 50--52°C, Fluka Chemie GmbH, Buchs,
Switzerland) and ferrofluid (APG 314, Ferrotec Inc., Santa Clara, CA,
USA) was loaded onto the ferrowax grooves of the top layer. The
assembled disc was finally laminated using a steel roller to eliminate
any gaps or air bubbles between the tape and polycarbonate plates. A
pressure-sensitive adhesive tape (HJ-Bioanalytik GmbH, Mönchengladbach,
Germany) was used to seal the inlet and outlet holes during the dilution
operation.

2.2. Rotor and optical systems for the centrifugal microfluidic disc

The rotor system and its operating software were purchased from Hanra
Precision Engineering (Incheon, Korea) and modified by our lab. The
system was composed of an 808 nm laser diode (TTG50TEC, Laser Solution
Korea, Ansan, Korea) for the ferrowax microvalve operation, a camera
(BFS-U3-16S2C-CS, FLIR Systems, Inc., Boston, MA, USA), and stroboscopic
lights (BUB 0641 (G), PerkinElmer, Inc., Waltham, MA, USA) for the
imaging of the disc. The laser module was automatically controlled by a
one-dimensional step motor (EzM-42S-A, Fastech, Bucheon, Korea), and a
polar coordinate system was used to precisely control the laser
irradiation position on the disc based on the rotational angle and
radial distance from the edge. A rotary servo motor (SGM7J-02A, Yaskawa
Electric Corp., Kitakyushu, Japan) was used to rotate the disc. Home and
trigger sensors (EE-SX671A, Omron Corp., Kyoto, Japan) were used to
accurately adjust the rotational position. A detailed illustration of
the rotor system is provided in Figure S2.

2.3. Materials and sample preparation

Ultrapure DNase/RNase-free DW (Bioneer\textsuperscript{®}, Daejeon,
Korea) was used in all experiments. Crystal violet and other chemicals
for the reaction buffer were purchased from Sigma-Aldrich Corp. (St.
Louis, MO, USA) and used without further purification.
\emph{Bst}polymerase and RNase inhibitor were purchased from New England
Biolabs Inc. (Beverly, MA, USA). Thermostable RNase H was purchased from
NanoHelix Co., Ltd. (Daejeon, Korea). All oligonucleotides used in the
study (Table S1) were synthesized and purified by Integrated DNA
Technologies, Inc. (Coralville, IA, USA). Outer primers were purified by
polyacrylamide gel electrophoresis (PAGE), while inner primers and
cycling probe technology (CPT) probes were purified by high-performance
liquid chromatography (HPLC). The target bacteria, \emph{Escherichia
Coli} O157:H7 were provided by Dxgene, Inc. (Seoul, Korea). The bacteria
were first cultivated at 37ºC for 16 h, and the colony-forming unit
(CFU) was measured using plate count agar (PCA) (NaraeBiotech, Inc.,
Gunpo, Korea). Based on the determined CFU, the culture solution was
diluted to 2.0 × 10\textsuperscript{8} CFU/mL, which was then
centrifuged at 11,000 rpm for 5 min (Centrifuge 5424, Eppendorf,
Hamburg, Germany). The resulting precipitant was resuspended in the same
volume of Tris-HCl buffer (10 mM, pH 8.8), which was then subjected to
the serial dilution on the centrifugal microfluidic disc.

2.4. Optical analysis and isothermal amplification

For the absorbance analysis of crystal violet, 40 \selectlanguage{greek}µ\selectlanguage{english}L of the finally
diluted crystal violet solutions were collected from the product
chambers and mixed with 120 \selectlanguage{greek}µ\selectlanguage{english}L of DW, which was then analyzed by
measuring the absorbance at 588 nm with a microplate reader (Infinite
M200 Pro, Tecan Group Ltd., Maennedorf, Switzerland).

For the real-time isothermal amplification, 50 \selectlanguage{greek}µ\selectlanguage{english}L of serially diluted
each bacterial sample was thermally lysed at 100 \selectlanguage{ngerman}°C for 10 min using a
heat block (MS-100, Hangzhou Allsheng Instruments Co., Ltd., Hangzhou,
China). Ten \selectlanguage{greek}µ\selectlanguage{english}L of the lysed bacterial sample was then mixed with 10 \selectlanguage{greek}µ\selectlanguage{english}L
of isothermal target and probe amplification (iTPA) {[}24, 25{]} reagent
solution to prepare the final reaction solution (20 \selectlanguage{greek}µ\selectlanguage{english}L) containing inner
primers (1 \selectlanguage{greek}µ\selectlanguage{english}M each), outer primers (0.1 \selectlanguage{greek}µ\selectlanguage{english}M each), dNTPs (1.6 mM each),
10 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4}, 10 mM
MgSO\textsubscript{4}, 0.8 mM spermine, 6 mM dithiothreitol, 5 ng/\selectlanguage{greek}µ\selectlanguage{english}L
bovine serum albumin, \emph{Bst} polymerase large fragment (0.15 U/\selectlanguage{greek}µ\selectlanguage{english}L),
RNase inhibitor (0.3 U/\selectlanguage{greek}µ\selectlanguage{english}L), thermostable RNase H (0.3 U/\selectlanguage{greek}µ\selectlanguage{english}L), and a 50 nM
CPT probe. The reaction solution was subjected to the iTPA reaction at
65\selectlanguage{ngerman}°C for 90 min using a CFX Connect\selectlanguage{english} Real-Time System (Bio-Rad
Laboratories, Inc., Richmond, CA, USA). A detail description of the iTPA
reaction is provided in Figure S3, or our previous reports {[}24, 25{]}.

\textbf{3. Results and discussion}

3.1. Design and operational principle of the centrifugal microfluidic
system

The centrifugal microfluidic disc designed in this work is composed of
six identical sections to allow six simultaneous dilution processes.
Each section mainly consists of two diluent chambers, a single dilution
chamber, seven product chambers, and connecting channels between them,
as shown in Figure 2(a). The diluent needs to be loaded to the two
identical diluent chambers prior to the operation, and the user needs to
apply only the target analyte solution to the dilution chamber to start
the serial dilution process. After the diluent and analyte solutions are
loaded through the green-colored inlet holes above the respective
chambers, the inlet and outlet holes are all sealed using a
pressure-sensitive adhesive tape. Each diluent chamber has three outlet
channels at different levels, such that the diluent chamber is radially
occupied by three equivalent volumes of the diluent. The six outlet
channels were initially all closed by the ferrowax microvalves (DV1
\textasciitilde{} DV6, diluent valve) to isolate the dilution chamber
from the diluent chambers. The main channel to elute the diluted sample
from the dilution chamber to the product chamber has seven initially
open ferrowax microvalves (EV1 \textasciitilde{} EV7, elution valve),
which is disconnected to the seven final product chambers by the closed
ferrowax microvalves (FV1 \textasciitilde{} FV7, final valve) located at
the entry of the seven respective branch channels.

The process begins by eluting the original 1 X analyte solution by
rotating the disc at 2,000 rpm after opening FV1. Then, predetermined
volume of 1 X solution was eluted to the first product chamber, while
the rest of the 1 X solution remained in the dilution chamber. For the
first dilution, EV1 was closed to isolate the 1 X product chamber from
the main channel, and DV1 was opened, followed by centrifugation at
2,000 rpm. Then, one third the initial volume of diluent was eluted to
the dilution chamber. For the complete mixing of the analyte solution
and the diluent, the disc was shaken by repeatedly rotating at 60
degrees clockwise and counterclockwise. After shaking, the first diluted
solution was eluted to the second product chamber by opening the FV2 and
rotating the disc at 2,000 rpm. The second and more consecutive
dilutions can be accomplished by repeating the same dilution procedure
but appropriately changing the microvalves to be opened and closed
(Figure 2(b)).

As illustrated in Figure S4, the ferrowax was loaded in the groove of
the top layer, which was then integrated with the bottom layer having
discontinuous deep channel and the middle layer having the channel part
peeled off. The ferrowax microvalves within the fabricated channels on
the disc were initially all in an open state, but the DVs and FVs were
made to be closed prior to the dilution process. The valve-closing
process was automatically executed by irradiating the laser to the
groove part to melt the ferrowax. Due to the capillary force, the melted
ferrowax would be guided to the nearby shallow channel, where the
ferrowax was solidified, consequently blocking the channel. Whenever
needed, the closed channels were reopened by irradiating the laser to
the shallow channel blocked by the ferrowax. The melted ferrowax was
removed away by flowing in the same direction with the solution.

3.2. Six consecutive serial two-fold dilutions for crystal violet
solution

To prove the serial dilution capability of the centrifugal microfluidic
system, we first fabricated the disc capable of conducting six
consecutive two-told serial dilutions and applied it for the serial
dilution of crystal violet solution using DW as a diluent. We used 256
\selectlanguage{greek}μ\selectlanguage{english}M solution of the crystal violet dye as an input 1 X concentration. To
start the dilution process, 100 \selectlanguage{greek}μ\selectlanguage{english}L of 1 X crystal violet solution and
150 \selectlanguage{greek}μ\selectlanguage{english}L of DW (300 \selectlanguage{greek}μ\selectlanguage{english}L for two chambers) were loaded to the dilution and
diluent chambers, respectively. To fully accommodate the solutions
during the dilution procedure, the three types of chambers were
fabricated to be slightly larger than the volumes actually needed by the
solutions (Figure 3(a)).

As presented in Figure 3(b), a single diluent chamber is supposed to be
radially occupied by the three equivalent 50 \selectlanguage{greek}μ\selectlanguage{english}L volumes of the 150 \selectlanguage{greek}μ\selectlanguage{english}L DW
diluent. As explained earlier, the six outlet channels from the diluent
chambers were initially all closed, which are serially opened to release
each 50 \selectlanguage{greek}μ\selectlanguage{english}L of the diluent from the diluent chamber to the dilution
chamber. The dilution chamber was configured to elute half the volume
(50 \selectlanguage{greek}μ\selectlanguage{english}L) of the initial solution or the diluted solutions by opening and
closing the appropriate ferrowax microvalves within the connecting
channels, followed by rotating the disc, while retaining the rest half
the volume (50 \selectlanguage{greek}μ\selectlanguage{english}L) in the dilution chamber. After the elution of the
initial 1 X sample solution, the first two-fold dilution can be
performed by the consecutive process of the diluent addition, mixing,
and elution. By repeating these dilution procedures six times (Figure
3(c)), we completed the six consecutive serial dilutions for the crystal
violet solution. Actual images of the initial and final disc are shown
in Figure 4, and step-by-step images of this process are provided in
Figure S5. A video is also available in the supporting information
(Movie S1).

After the completion of the serial dilutions, we collected the diluted
crystal violet solutions (50 \selectlanguage{greek}μ\selectlanguage{english}L each) from the final seven product
chambers, which range from 1 X (256 \selectlanguage{greek}μ\selectlanguage{english}M) to 2\textsuperscript{-6} X (4
\selectlanguage{greek}μ\selectlanguage{english}M), and analyzed them by spectrophotometer. Based on the spectrum from
the samples, (Figure 4(c)), the absorbances at 588 nm were obtained and
plotted against the concentrations of the diluted samples (Figure 4(d),
red data) using the least square method. As a result, the curve showed
excellent linearity (R\textsuperscript{2} [?] 0.9910), quite comparable
with that from the manual pipetting (Figure 4(d), black data), clearly
verifying the accurate dilution capability of this centrifugal
microfluidic system.

3.3. Six consecutive serial ten-fold dilutions for bacterial sample

To further verify the versatile capability of the system for the
dilution in a wide dynamic range, we next fabricated the centrifugal
microfluidic disc capable of accomplishing six consecutive ten-fold
serial dilutions and applied it for the dilutions of the biological
sample containing \emph{Escherichia coli} O157:H7. As presented in
Figure 5(a), the overall structural elements were all the same with the
two-fold dilution system, and the same top layer used for two-fold
dilution was used for ten-fold dilution. The principal design of the
diluent chamber in the bottom layer was also almost same with that for
two-fold dilution, and the each diluent chamber was designed to be
radially occupied by three one third the volumes of the diluent (Figure
5(b)). However, the volumetric structures of the chambers in the bottom
layer were modified to suit ten-fold dilutions. More specifically, the
serially added diluent volume was changed to 90 \selectlanguage{greek}µ\selectlanguage{english}L from 50 \selectlanguage{greek}µ\selectlanguage{english}L in
two-fold dilution, and the volume of the diluent chamber was accordingly
larger to accommodate the larger volume of the diluent. The volumetric
design of the dilution chamber was also quite modified, such that 90 \%
of the diluted samples (90 \selectlanguage{greek}µ\selectlanguage{english}L) were eluted to the product chambers while
leaving only the rest 10 \% of the samples (10 \selectlanguage{greek}µ\selectlanguage{english}L).

In this manner, the dilution ratio could be manipulated simply by
changing the serially added diluent volume from the diluent chamber and
the remaining analyte volume in the dilution chamber. To start the
ten-fold dilution process, 100 \selectlanguage{greek}µ\selectlanguage{english}L of the target bacterial sample and 270
\selectlanguage{greek}µ\selectlanguage{english}L of Tris-HCl buffer diluent (540 \selectlanguage{greek}μ\selectlanguage{english}L for two chambers) were loaded into
the respective dilution and diluent chambers. Again, to conveniently
accommodate the solutions during the dilution procedure, the three types
of chambers were fabricated to be slightly larger than the volumes
actually needed by the solutions. \emph{Escherichia coli} O157:H7 sample
of 2.0 \selectlanguage{ngerman}× 10\textsuperscript{8} CFU/mL was set as a 1 X sample, and it
was ten-fold serially diluted down to 2.0 × 10\textsuperscript{2} CFU/mL
following the same procedures for two-fold dilutions (Figure 5(c)).
Actual images of the initial and final disc are shown in Figure 6, and
step-by-step images of this process are provided in Figure S6.

After the completion of the six consecutive serial dilutions, the
diluted bacterial samples were collected from the final seven product
chambers, and mixed with iTPA reagent solution to prepare the final
seven reaction solutions ranging from 1.0 × 10\textsuperscript{2} to 1.0
× 10\textsuperscript{8} CFU/mL. To validate the accuracy of the
automated serial dilutions in such a wide dynamic range, we performed
iTPA reactions for the seven samples and obtained the real-time
fluorescence curves from the amplified bacterial DNAs (Figure 6(c)).
Based on the curves, we determined threshold time (T\textsubscript{t}),
defined as the reaction time at which the fluorescence signal exceeds
the threshold line (fluorescence intensity = 120) for the seven target
concentrations, which was then plotted against the log of target DNA
concentration (Figure 6(d), red data) using the least square method. As
a result, the curve showed excellent linearity (R\textsuperscript{2} \selectlanguage{english}[?]
0.9818) in a range from 1.0 x 10\textsuperscript{2} to 1.0 x
10\textsuperscript{7} CFU/mL, confirming the accurate dilution
capability of the centrifugal microfluidic system, which is almost the
same with that (R\textsuperscript{2} \selectlanguage{english}[?] 0.9815) of the conventional
manual pipetting (Figure 6(d), black data). Unfortunately, the
T\textsubscript{t} value from the sample of 1.0 x
10\textsuperscript{8}CFU/mL deviated from the line. We assume that the
lysed cell debris and other inhibiting components from the bacterial
sample at such a high concentration of 1.0 x 10\textsuperscript{8}
CFU/mL might have impeded the iTPA reaction because we conducted the
iTPA reaction directly using the lysed samples without any prior
purification of nucleic acids. However, this deviation of the initial
sample is just due to the limitation of the dynamic range of the iTPA
technique but not associated with dilution process. All these results
confirm that the developed centrifugal microfluidic system is quite
capable of accurately diluting the samples in a very wide dynamic range,
up to six orders of magnitude.

\textbf{4. Conclusion}

In this study, we developed a new centrifugal microfluidic system
capable of accomplishing fully automated serial dilutions, by utilizing
ferrowax microvalves. Using the centrifugal microfluidic system, we
successfully achieved six consecutive two-fold and ten-fold dilutions
for the crystal violet solution and the \emph{Escherichia coli} O157:H7
sample, respectively, in a very accurate and loss-free manner. Very
importantly, this system was fully automated by taking advantage of the
automated actuation of the ferrowax microvalves such that any handling
step was not required after the initial loading of the analyte.
Moreover, the overall process was completed very rapidly within five
minutes due to the elegantly designed minimized procedures. We believe
that the fully automated system developed herein has a great potential
for the development of diverse point-of-care diagnostic systems
involving the serial dilution process.

\textbf{Supporting Information}

Supporting Information is available from the Wiley Online Library or
from the author.

\textbf{Acknowledgements}

This work was supported by the Industrial Technology Innovation Program
(20005957) funded by the Ministry of Trade, Industry \& Energy (MI,
Korea), the Technology Development Program (S2735682) funded by the
Ministry of SMEs and Startups (MSS, Korea), and the BioNano Health-Guard
Research Center funded by the Ministry of Science and ICT (MSIT) of
Korea as Global Frontier Project (H-GUARD\_2013M3A6B2078964).

\textbf{Conflict of interest}

The authors declare no conflict of interest.

\textbf{References}

{[}1{]} J.X. Shen, C.I. Tama, R.N. Hayes, \emph{J. Chromatogr. B Analyt.
Technol. Biomed. Life Sci.} \textbf{2006} , \emph{843} , 275.

{[}2{]} Z. Ouyang, S. Federer, G. Porter, C. Kaufmann, M. Jemal,\emph{J.
Assoc. Lab. Autom.} \textbf{2008} , \emph{13} , 24.

{[}3{]} R.E. Jones, W. Zheng, J.C. McKew, C.Z. Chen, \emph{J. Lab.
Autom.} \textbf{2013} , \emph{18} , 367.

{[}4{]} K.P. Smith, J.E. Kirby, \emph{J. Clin. Microbiol.}\textbf{2016}
, \emph{54} , 2288.

{[}5{]} S. Steffens, L. Nusser, T.B. Seiler, N. Ruchter, M. Schumann, R.
Doring, C. Cofalla, A. Ostfeld, E. Salomons, H. Schuttrumpf, H. Hollert,
M. Brinkmann, \emph{PLoS One} \textbf{2017} , \emph{12} , e0179636.

{[}6{]} M.C. Kong, E.D. Salin, \emph{Anal. Chem.} \textbf{2012}
,\emph{84} , 10038.

{[}7{]} Y. Ouyang, J. Li, C. Phaneuf, P.S. Riehl, C. Forest, M. Begley,
D.M. Haverstick, J.P. Landers, \emph{Lab Chip} \textbf{2016} , \emph{16}
, 377.

{[}8{]} W.B. Lee, C.Y. Fu, W.H. Chang, H.L. You, C.H. Wang, M.S. Lee,
G.B. Lee, \emph{Biosens. Bioelectron.} \textbf{2017} , \emph{87} , 669.

{[}9{]} K.H. Shinji Sugiura, Toshiyuki Kanamori, \emph{Anal.
Chem.}\textbf{2010} , \emph{82} , 8278.

{[}10{]} S. Ahrar, M. Hwang, P.N. Duncan, E.E. Hui,
\emph{Analyst}\textbf{2014} , \emph{139} , 187.

{[}11{]} L. Derzsi, T.S. Kaminski, P. Garstecki, Antibiograms in five
pipetting steps: precise dilution assays in sub-microliter volumes with
a conventional pipette, \emph{Lab Chip} \textbf{2016} , \emph{16} , 893.

{[}12{]} N. Zijlstra, F. Dingfelder, B. Wunderlich, F. Zosel, S. Benke,
D. Nettels, B. Schuler, \emph{Angew. Chem. Int. Ed. Engl.}\textbf{2017}
, \emph{56} , 7126.

{[}13{]} J. Ducree, S. Haeberle, S. Lutz, S. Pausch, F.v. Stetten, R.
Zengerle, \selectlanguage{english}\emph{J. Micromech. Microeng.} \textbf{2007} , \emph{17} ,
S103.

{[}14{]} H. Hwang, S.H. Kim, T.H. Kim, J.K. Park, Y.K. Cho, \emph{Lab
Chip} \textbf{2011} , \emph{11} , 3404.

{[}15{]} C. Escadafal, O. Faye, A.A. Sall, O. Faye, M. Weidmann, O.
Strohmeier, F.v. Stetten, J. Drexler, M. Eberhard, M. Niedrig, P.
Patel,\emph{PLoS Negl. Trop. Dis.} \textbf{2014} , \emph{8} , e2730.

{[}16{]} S.J. Oh, B.H. Park, J.H. Jung, G. Choi, D.C. Lee, D.H. Kim, T.
S. Seo, \emph{Biosens. Bioelectron.} \textbf{2016} , \emph{75} , 293.

{[}17{]} B.H. Park, S.J. Oh, J.H. Jung, G. Choi, J.H. Seo, D.H. Kim,
E.Y. Lee, \emph{Biosens. Bioelectron.} \textbf{2017} , \emph{91} , 334.

{[}18{]} A. Sayad, F. Ibrahim, S. Mukim Uddin, J. Cho, M. Madou, K.L.
Thong, \emph{Biosens. Bioelectron.} \textbf{2018} , \emph{100} , 96.

{[}19{]} I. Maguire, R. O'Kennedy, J. Ducree, F. Regan, \emph{Anal.
Methods} \textbf{2018} , \emph{10} , 1497.

{[}20{]} H.K. Woo, J. Park, J.Y. Ku, C.H. Lee, V. Sunkara, H.K. Ha, Y.K.
Cho, \emph{Lab Chip} \textbf{2018} , \emph{19} , 87.

{[}21{]} T.-H. Kim, C.-J. Kim, Y. Kim, Y.-K. Cho, \emph{Sens. Actuators
B Chem.} \textbf{2018} , \emph{256} , 310.

{[}22{]} P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, M. Keller,
T. Hutzenlaub, F.v. Stetten, R. Zengerle, N. Paust, \emph{Lab
Chip}\textbf{2019} , \emph{19} , 2205.

{[}23{]} J.M. Park, Y.K. Cho, B.S. Lee, J.G. Lee, C. Ko, \emph{Lab
Chip}\textbf{2007} , \emph{7} , 557.

{[}24{]} C. Jung, U.O. Kim, M.H. Kim, H.G. Park, \emph{Anal.
Chem.}\textbf{2010} , \emph{82} , 5937.

{[}25{]} C. Jung, J.W. Chung, U.O. Kim, M.H. Kim, H.G.
Park,\emph{Biosens. Bioelectron.} \textbf{2011} , \emph{26} , 1953.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

