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Methods in
Molecular Biology 2027
Jessica E. Fitzgerald
Hicham Fenniri Editors
Biomimetic
Sensing
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
Edited by
Jessica E. Fitzgerald
Departments of Bioengineering and Chemical Engineering, Northeastern University, Boston, MA, USA
Hicham Fenniri
Departments of Chemical Engineering, Bioengineering, Chemistry and Chemical Biology, Northeastern
University, Boston, MA, USA
Editors
Jessica E. Fitzgerald Hicham Fenniri
Departments of Bioengineering Departments of Chemical Engineering, Bioengineering,
and Chemical Engineering Chemistry and Chemical Biology
Northeastern University Northeastern University
Boston, MA, USA Boston, MA, USA
Cover illustration: Barcoded polymer-based cross-reactive sensor array response to a mixture of analytes. The different
colors illustrate different responses from the sensory elements.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface
The Gap in Technology: Fast, Facile, and Quantifiable Detection of Analytes in Vapor
and Liquid [1, 2]
Detection of analytes for vapor and liquid deconvolution has been performed in a variety of
fields, including nutrition, toxicology, biomedicine, and chemistry. The gold standard for
vapor sensing is gas chromatography-mass spectrometry (GC-MS), which is a quantitative
sample analysis that provides both the type and amount of analytes present in a sample,
usually in the form of volatile organic compounds (VOCs). While it is advantageous to know
which specific VOCs are present in a sample, GC-MS is not practical for widespread use in
vapor sensing because it requires both specialized, expensive equipment and highly trained
personnel for operation. In addition, some relevant data may be lost due to vapor
pre-concentration and sampling techniques. Similarly, the most common liquid sensing
techniques require sample labeling, or tagging, before performing an assay. This sensing
technique is limited because each analyte requires a specific label (making multiplexed
sensing difficult), the analytes in the sample must be known beforehand so that the correct
label can be selected, and nonspecific binding may take place, affecting the accuracy of the
measured analyte concentration. As sensing needs to continue to expand and develop, there
remains a need for methods that enable fast, facile, and accurate sensing at a low cost. To
meet these needs, many researchers have looked to the mammalian olfactory system as a
model for label-free, multiplexed sensing, developing platforms that are easy to use and
produce and that can be used in a wide variety of applications.
There are about 1000 genes that encode olfactory receptors (ORs), and each OR has
multiple sites for odorant binding, enabling the detection of more than one odorant for
each OR, a characteristic called cross-reactivity. Different combinations of activated recep-
tors make up unique signaling codes, or “fingerprints,” for specific odorants, making it
possible to distinguish between thousands. This sensing platform has inspired researchers
over the past several decades to develop sensing devices that are cross-reactive and accurate
and have multiplexing capabilities. These biomimetic devices are called “electronic/artificial
noses” (e-noses) or e-tongues to detect certain analytes present in both vapors and liquids,
respectively. E-devices have proved to be successful in a broad range of scientific and
engineering fields, providing cost-effective, minimally invasive (in the case of clinical use),
and highly accurate vapor and liquid component analysis. In this book, we highlight the
potential of e-device technology to serve as a successful platform for multiplexed sensing,
along with the methods for device fabrication, calibration, and assays in multiple applica-
tions. The subsequent sections describe e-device sensing platforms, explore their use, and
outline existing limitations and future directions in device development.
v
vi Preface
The first use of the term “electronic nose” was at a conference in 1987, and the first
conference dedicated specifically to artificial olfaction was in 1989. Gardner and Bartlett
originally defined an “electronic nose” as follows: “an instrument, which comprises of an
array of electronic-chemical sensors with partial specificity and an appropriate pattern recog-
nition system, capable of recognising simple or complex odours.” The first devices that utilized
this technology were comprised of sets of distinct active materials, each connected to its own
signal transduction channel. When passing an analyte vapor over the sensor array, activated
sensors would transmit an electrical signal to a processor, which would then alert the user of
the analytes present by cross-referencing the list of activated sensors with a database of
known analyte profiles. The earliest designs employed metal oxide semiconducting field
effect transistors (MOSFETs) as electrical sensors to detect gases such as NO2. This was
based on the principle that the conductivity of semiconductor metals changes upon variance
in the atmospheric gas surrounding the sensor. MOSFETs are usually constructed from a
SiO2 insulating layer, with a semiconductor metal deposited on top as the gate in the circuit.
A voltage is applied to maintain a constant current, and as the gas adsorbs onto the gate, the
conductance of the FET changes, thereby causing the voltage to change. e-devices are able
to differentiate analytes via “fingerprint” outputs; that is, each sample’s analyte profile
(comprised of a unique mixture of analyte types and relative concentrations) produces a
unique response pattern from the sensor array, enabling sample differentiation. New sample
fingerprints are compared to known sample fingerprints via data analysis (in most cases
multivariate data analysis such as principle component analysis), which enables pattern
recognition, clustering, and classification of the unknown analyte sample.
This fingerprint sensing method has been implemented in a number of fields, including
food science, quality control, drug testing, contamination detection, defense efforts (e.g.,
explosive detection), and medical diagnosis. Some of the most pioneering work has been
done in medical diagnostics via exhaled breath analysis. Many metabolites, or metabolic
by-products, have been identified and correlated with specific diseases; some of these can
even be a good indicator of disease progression. Many of these metabolites are volatile
organic compounds (VOCs), which are small molecules that enter exhaled breath through
gas exchange at the alveolar-capillary membrane of the respiratory tract. While the VOCs
produced in each disease are thought to be primarily from oxidative stress, the subsequent
effect of each disease on the body is unique and leads to the production of disease-specific
VOC profiles. A reliable, noninvasive device capable of detecting subtle molecular changes
and differences can be leveraged to implement this personalized medicine approach. The
e-devices included in this work have much potential for implementation in these fields and
others, as their sensitivity and specificity can be tuned toward specific analytes of interest.
Within the past several years, researchers have taken advantage of rapid technological
advances to expand and improve e-device sensing platforms and sensor materials, signal
transduction mechanisms, and data processing for pattern recognition and analyte identifi-
cation. Moreover, as more became known about the physiology of the mammalian olfactory
system, sensors further advanced to take advantage of these new findings, from
Preface vii
incorporating sensors that were able to detect multiple analytes to the development of a
biomimetic flow chamber to enhance analyte detection at low levels. This allowed for a
greater number of analytes to be recognized using a smaller array of multi-selective sensors.
Within the last two decades, electronic tongues for liquid detection have also been devel-
oped, mimicking the olfactory signaling pathways.
The types of sensors employed for both vapor and liquid samples in these devices vary,
including gravimetric, or mass, electrochemical, and optical sensors, allowing characteriza-
tion of analytes based on mass, electrical properties (e.g., conductance impedance), and
electron/photon interactive properties, respectively. Gravimetric sensors are either piezo-
electric (PZ) crystals or microcantilevers, which have a specific resonant frequency. On
binding with an analyte, the resonant frequency of the sensor drops in proportion to the
added mass, due to either viscoelastic or gravimetric effects. Electrochemical e-devices are
comprised of an electronic circuit connected to a network of sensory materials—most
commonly conductive polymers or metal oxides—that provide an electrical response on
binding with a specific known analyte. This response is characterized by monitoring sensor
conductivity, resistivity, or voltage change during vapor exposure. Finally, optical sensors
work by displaying a shift in the emission or absorption of different types of electromagnetic
radiation on binding with a desired analyte. There are two popular means of optical
detection: fluorescent sensors, which fluoresce upon analyte binding, and colorimetric
sensors, which display a visible color change upon analyte binding.
Although gravimetric sensors have proved to be successful, there are many limitations
with this device setup. Inaccuracies due to subtle changes in surface coating, humidity, or
temperature necessitate frequent calibrations, which is unfortunately delicate and time-
consuming. e-devices that employ optical and electrochemical sensors have shown much
promise as they provide an easier and more cost-effective way of identifying analytes in vapor
while maintaining accuracy. Optical sensors offer significant benefits compared with those
mentioned above since they can provide multiple complex data types simultaneously,
including changes in intensity, fluorescence lifetime, wavelength, and spectral shape. This
approach increases the ratio of recognizable analytes to the number of sensors used. This
work mainly focuses on optical (Chapters 1–10) and electrochemical (Chapters 11–13)
methods of sensing, as these are at the forefront of e-device technology, being more stable
and reliable than their gravimetric counterparts. We have also included a chapter on a
cutting-edge mechanochemical sensing method using folded DNA origami structures
(Chapter 14) that have been demonstrated to have a limit of detection down to the single
molecule level. Finally, we highlight here some cutting-edge methods to optimize e-device
data and technology via drift correction and calibration (Chapter 15) and computer model-
ing of sensor output for material optimization (Chapter 16).
Optical sensors in e-device systems have shown much promise to provide a facile, cost-
effective, and accurate way of identifying analytes in vapor and liquid samples. Optical
sensors display a shift in emission or absorption of different types of radiations upon analyte
binding. The two most popular means of detection are spectroscopic and colorimetric
sensing. In this work, we highlight the methods and applications of both means of detec-
tion. The spectroscopic methods employ Raman spectroscopy (Chapter 1), interferometry
(Chapter 2), mass spectrometry (Chapter 3), fluorescence microscopy (Chapter 5), and
viii Preface
surface plasmon resonance (Chapters 8 and 10). The colorimetric sensors herein are pro-
duced in several ways, including microchip fabrication and photolithography (Chapters 4, 6,
and 7). As mentioned previously, optical sensors offer significant benefits compared with
gravimetric sensors; they can provide multiple complex data types simultaneously, including
changes in intensity, fluorescence and colorimetric lifetime, wavelength, and spectral shape.
This increases the ratio of recognizable analytes to the number of sensors used. In addition,
microsphere optical arrays such as those developed by Walt et al. and presented in Chapter 1
provide an advantage over other multisensor systems because billions of beads that produce
an identical response can be made simultaneously, compared with many sensors for which
the fabrication process is tedious. Each type of bead has a distinct, intrinsic response to the
samples presented, which eliminates the need for additional encoding for bead
identification.
As e-device implementation continues to grow in breadth, there are certain limiting factors
that must be addressed. First, there remains a lack of standards for sample collection, both
environmental (ambient air, water) and medical (exhaled breath, saliva). When developing a
sampling method, it is important to optimize the collection, preparation, and storage
method to maximize analyte detection without denaturing or altering the chemical profile
of the sample. For example, collecting alveolar breath, the second phase of the breathing
cycle, requires a sampling method that minimizes VOC interference from ambient air while
capturing the alveolar air from a patient who is breathing steadily at a set velocity. The
sample storage material and time of storage also affect analyte recovery. Even after obtaining
an ideal sample, e-device performance accuracy may be limited by extrinsic factors such as
humidity and temperature or intrinsic factors such as sensor drift and instrumentation
errors. Additionally, e-device analyte fingerprint analysis via pattern recognition requires
complex data analysis, which currently limits the widespread implementation of these
devices.
Preface ix
Finally, though preliminary studies with e-devices have been largely successful, their
reproducibility is limited because methods must be optimized de novo for each specific
application. Standards need to be developed from statistical analysis of device performance
and should include thresholds for success in areas such as response reproducibility, specific-
ity, and sensitivity. In developing these standards, it is also important to consider the
ultimate goal of the device. For example, if the goal is simply to diagnose and classify a
sample, such as one that correlates with a specific disease, selectivity is more important than
sensitivity; however, if the goal is to monitor analyte profile change over time, such as with
disease progression, sensitivity to slight variations in analyte concentrations is of great
importance.
Though these limitations currently serve as a bottleneck for widespread e-device imple-
mentation, researchers continue to work diligently to develop methods that will circumvent
and overcome them. For example, Chapter 15 includes a detailed method for device
calibration that can be applied to a wide variety of sensing mechanisms and e-devices.
Additionally, complementary methods, such as computer modeling of analyte-sensor inter-
action presented in Chapter 16, serve as a way to better predict sensor response to specific
analytes. This can then be leveraged to produce devices that are highly tuned toward a
specific analyte or group of analytes, i.e., much improved sensitivity and selectivity. As the
technology for e-device sensing continues to trend toward portable, accurate, and easy-to-
use platforms, they have great potential to be implemented wherever analyte detection is
required. Indeed, they may be able to reduce the need for highly specialized, expensive
equipment and personnel. For developing countries in particular, e-devices that are simul-
taneously inexpensive may soon be able to take the place of highly specialized equipment in
fields such as defense efforts and explosive detection, water and food contamination, and
personalized medicine.
References
1. Fitzgerald JE, Bui ETH, Simon NM, Fenniri H (2016) Trends Biotechnol. 1
2. Fitzgerald JE, Fenniri H (2016) RSC Adv. 6(84): 80468
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
xi
xii Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Contributors
NAGEH K. ALLAM Energy Materials Laboratory, School of Sciences and Engineering, The
American University in Cairo, New Cairo, Egypt
MARIA L. BRAUNGER Department of Applied Physics, “Gleb Wataghin” Institute of Physics
(IFGW), University of Campinas—UNICAMP, Campinas, SP, Brazil
COLLIN J. BRIGHT U.S Army Combat Capabilities Development Command, C5ISR Center,
Fort Belvoir, VA, USA
OLALLA CALVO-LOZANO Nanobiosensors and Bioanalytical Applications Group
(NanoB2A), Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and
BIST, Barcelona, Spain; Networking Center on Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Barcelona, Spain
DANIEL CITTERIO Faculty of Science and Technology, Department of Applied Chemistry,
Keio University, Yokohama, Japan
CRISTIANE M. DAIKUZONO Centro de Ciências e Tecnologias para Sustentabilidade (CCTS),
Universidade Federal de São Carlos—UFSCar, Sorocaba, SP, Brazil
ADIL DENIZLI Department of Chemistry, Hacettepe University, Ankara, Turkey
MARI C. ESTÉVEZ Nanobiosensors and Bioanalytical Applications Group (NanoB2A),
Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST,
Barcelona, Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Barcelona, Spain
HICHAM FENNIRI Departments of Chemical Engineering, Bioengineering, Chemistry and
Chemical Biology, Northeastern University, Boston, MA, USA; Department of
Bioengineering, Northeastern University, Boston, MA, USA
JESSICA E. FITZGERALD Department of Bioengineering and Department on Chemical
Engineering, Northeastern University, Boston, MA, USA
M. TERESA S. R. GOMES Chemistry Department, University of Aveiro, Aveiro, Portugal;
CESAM, University of Aveiro, Aveiro, Portugal
VERA L. V. GRANADO Chemistry Department, University of Aveiro, Aveiro, Portugal
YUMA IKEDA Faculty of Science and Technology, Department of Applied Chemistry, Keio
University, Yokohama, Japan
A. T. CHARLIE JOHNSON Department of Physics and Astronomy, University of Pennsylvania,
Philadelphia, PA, USA
SAGUN JONCHHE Department of Chemistry and Biochemistry, Kent State University, Kent,
OH, USA
DMITRY KIRSANOV Institute of Chemistry, St. Petersburg State University, St. Petersburg,
Russia; Laboratory of Artificial Sensory Systems, ITMO University, St. Petersburg, Russia
LAURA M. LECHUGA Nanobiosensors and Bioanalytical Applications Group (NanoB2A),
Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST,
Barcelona, Spain; Networking Center on Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Barcelona, Spain
ANDREY LEGIN Institute of Chemistry, St. Petersburg State University, St. Petersburg,
Russia; Laboratory of Artificial Sensory Systems, ITMO University, St. Petersburg, Russia
FENGYU LI Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of
Sciences (ICCAS), Beijing Engineering Research Center of Nanomaterials for Green
xiii
xiv Contributors
YANLIN SONG Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy
of Sciences (ICCAS), Beijing Engineering Research Center of Nanomaterials for Green
Printing Technology, Beijing National Laboratory for Molecular Sciences (BNLMS),
Beijing, P. R. China
M. SELIM U€ NLÜ Department of Biomedical Engineering, Boston University, Boston, MA,
USA; Department of Electrical and Computer Engineering, Boston University, Boston,
MA, USA
NESE LORTLAR U € NLÜ Department of Biomedical Engineering, Boston University, Boston,
MA, USA
CELALETTIN YURDAKUL Department of Electrical and Computer Engineering, Boston
University, Boston, MA, USA
Chapter 1
Abstract
The development of chemical sensors continues to be an active area of research, especially the development
of a practical electronic nose. Here, we present a spectroscopic chemical sensor based on an array of 64 self-
encoded polymer films deposited on a microfabricated silicon substrate. The polymer arrays were analyzed
by FTIR and Raman spectroscopy before and after exposure to a series of organic volatiles to monitor
changes in their vibrational fingerprints. We show here that the spectroscopic changes of self-encoded
polymer films can be used to distinguish between volatile organic analytes. Changes induced in the sensor
arrays by the analyte vapor were denoted by a spectroscopic response of the self-encoded polymer sensors
and transformed into a response pattern by multivariate data analysis using partial least squares regression.
The results indicated that the polymer sensors provide a unique and reproducible pattern for each analyte
vapor and can potentially be used in the fabrication of a novel electronic nose device.
Key words Barcoded polymer, Sensor array, Raman spectroscopy, Multivariate data analysis
1 Introduction
Jessica E. Fitzgerald and Hicham Fenniri (eds.), Biomimetic Sensing: Methods and Protocols, Methods in Molecular Biology,
vol. 2027, https://doi.org/10.1007/978-1-4939-9616-2_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
1
2 Jessica E. Fitzgerald and Hicham Fenniri
2 Materials
1. Suspension copolymerization:
(a) Benzoyl peroxide (BPO).
(b) Poly(vinylalcohol) (PVA).
(c) 80% Divinylbenzene (DVB).
(d) Chloromethylstyrene.
(e) Co-monomers, distilled under reduced pressure to remove
the stabilizers and then stored at +4 C: styrene,
2,5-dimethylstyrene, 4-methylstyrene, 2,4-dimethylstyrene,
4-t-butylstyrene, 3-methylstyrene (see Note 1).
(f) The following solvents, distilled prior to use: N,N-
dimethylformamide (DMF), dichloromethane (DCM),
and toluene were distilled from CaH2; methanol and
ethanol from Mg, and tetrahydrofuran (THF) from
Na/benzophenone.
(g) Polypropylene woven mesh (0.003000 opening width).
(h) An IKA–RW20 mechanical motor.
(i) The reaction vessels and impellers were designed accord-
ing to the literature and shown in Fig. 1 [20]. A piece of
Pyrex tube of ca. 70 mm external diameter and 3–4 mm
thickness was placed in a glass working lathe, and by the
use of a specially made metal jig four equally spaced inden-
tations (baffles) were pressed along its length. After
annealing, the extension of a B40 Pyrex socket was joined
to one end of the tube, and two B19 sockets were then
attached to either side of the B40 socket. A B10 socket was
precisely positioned at one side of one of the indentations
at the head of the vessel. The other end of the tube was
then smoothly rounded off and the whole vessel annealed.
(j) Standard Morton flask (ChemGlass).
4 Jessica E. Fitzgerald and Hicham Fenniri
Fig. 1 Suspension polymerization apparatus. (a) and (b) are accession points for a reflux condenser and
nitrogen inlet, (c) is a sampling area, (d) is a stirrer guide, (e) is the stirrer with its position indicated for full-
scale operation. (f) is an expanded drawing of a single stirrer blade indicating the curvature at both ends and
the position and angle of attachment to the stirrer rod. All dimensions are in mm. Used with permission
from [20]
3 Methods
Fig. 2 Molecular structure of seven different monomers used for the fabrication
of the barcoded copolymers. Used with permission from [18]
6 Jessica E. Fitzgerald and Hicham Fenniri
Table 1
Weight percent of styrene monomers used for the synthesis of 25 BCR sensors
(f) Wash the beads with dH2O and ethanol using a Soxhlet
extractors (24 h each).
(g) Finally, sieve the beads and dry under vacuum.
2. Sensor array fabrication: Lithography and anisotropic dry reac-
tive ion etching (RIE) (see Note 6).
Cross-Reactive Polymer Film Arrays for Sensor Applications 7
(a) Piranha clean the silicon substrates and prime with HMDS
primer.
(b) Spin-coat with AZ P4620 photoresists.
(c) Expose areas of the silicon by irradiating the film
(λ ¼ 400 nm) through a lithography mask.
(d) Etch the exposed silicon wafer with RIE using a combina-
tion of SF6/C2F4, at pressures of 125 and 75 sccm,
respectively. In this way, 10, 100, and 200 μm deep wells
can be obtained.
(e) Coat the patterned wafer with a 200 μm thick copper film
by RF sputtering.
3. Polymer array deposition (see Notes 7 and 8):
(a) Deposit the polymer beads onto the micro-patterned sili-
con substrates by dusting an amount of beads on the
surface of the silicon chip.
(b) Use a rubberized spatula to spread the powder in a circular
motion over the chip to ensure the filling of the holes.
SEM of the BCR arrays (Fig. 3) clearly shows that by
Fig. 3 SEM images of etched micro-well (a), full substrate (b), deposited BCR bead (c), and complete sensor
array (d)
8 Jessica E. Fitzgerald and Hicham Fenniri
N2
Inlet Microscope Lens
Analyte Outlet
Sensor Arrays
Fig. 4 Schematic diagram of the analyte delivery setup and analyzing system for
recording response of the sensor array to the application of analyte (general
setup of the experiment). Exposure to a given analyte vapor induces changes in
the (IR or Raman) spectrum that are recorded. Used with permission from [7]
Fig. 5 FTIR (left) and Raman (right) of BP (3-methylstyrene/4-t-butylstyrene) before and after methanol vapor
exposure. Methanol and difference spectra between “before” and “after” methanol vapor. Used with
permission from [18]
10 Jessica E. Fitzgerald and Hicham Fenniri
y i ∗y i 0
cosθ ¼ ð1Þ
yi yi0
4 Notes
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14:767–770. https://doi.org/10.1016/j. graft copolymers. J Am Chem Soc
cbpa.2010.06.181 125:10546–10560. https://doi.org/10.
17. Fenniri H, Chun S, Terreau O, Bravo-Vasquez 1021/ja035665q
JP (2008) Preparation and infrared/Raman
Chapter 2
Abstract
The determination of kinetic information and appropriate binding pairs is fundamental to the proper
optimization and selection of ligands used in immunoassays, diagnostics, and therapeutics. However, the
ability to estimate such parameters in a multiplexed and inexpensive format remains difficult and modifica-
tion of the ligand is often necessary. Here, we detail the methods and materials necessary to evaluate
hundreds of unlabeled ligands simultaneously using the interferometric reflectance imaging sensor (IRIS).
The incorporation of a low-cost fluidic cartridge that integrates on the top of the sensor simplifies reagent
handling considerably.
Key words Interferometric reflectance imaging sensor (IRIS), Multiplexed kinetics, Label-free bind-
ing, Dengue NS1
1 Introduction
Jessica E. Fitzgerald and Hicham Fenniri (eds.), Biomimetic Sensing: Methods and Protocols, Methods in Molecular Biology,
vol. 2027, https://doi.org/10.1007/978-1-4939-9616-2_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019
15
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peitossa oli tuo tulija. Ei, nyt se vihreä hävisi kokonaan ja sen sijaan
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Piksa! Piksahan se olikin.
PIRUNKAUKALO
Hän puristi lujasti huulensa yhteen ja veti airoilla, niin että vesi
veneen kokassa kohisi.
— Eivät muut vielä ehtineet kuin Piksa ja Pusu. Mutta kyllä minä
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sen kummempaa ollut, korkeita kallioita vain.
Aamos söi, sillä hänellä oli aika nälkä. Mutta syödessään hän
ajatteli monenlaisia asioita. Ja aina kiertyivät ajatukset lopulta tähän
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Iso musta mies kivääri selässä. Sillä oli varmasti pahoja aikeita,
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