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Ka-Meng Lei · Pui-In Mak
Man-Kay Law · Rui Paulo Martins

Handheld
Total Chemical
and Biological
Analysis Systems
Bridging NMR, Digital Microfluidics, and
Semiconductors
Handheld Total Chemical and Biological
Analysis Systems
Ka-Meng Lei • Pui-In Mak
Man-Kay Law • Rui Paulo Martins

Handheld Total Chemical

and Biological Analysis
Systems
Bridging NMR, Digital Microfluidics,
and Semiconductors
Ka-Meng Lei Pui-In Mak
State-Key Laboratory of Analog State-Key Laboratory of Analog
and Mixed-Signal VLSI and Mixed-Signal VLSI and FST-ECE
University of Macau University of Macau
Macau, China Macau, China

Man-Kay Law Rui Paulo Martins

State-Key Laboratory of Analog State-Key Laboratory of Analog and
and Mixed-Signal VLSI Mixed-Signal VLSI and FST-ECE
University of Macau University of Macau
Macau, China Macau, China
Instituto Superior Técnico Universidade de
Lisboa
Lisbon, Portugal

ISBN 978-3-319-67824-5    ISBN 978-3-319-67825-2 (eBook)

DOI 10.1007/978-3-319-67825-2

Library of Congress Control Number: 2017952196

© Springer International Publishing AG 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
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in published maps and institutional affiliations.

Printed on acid-free paper

This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

This book investigates the handheld total chemical and biological analysis system
implemented with complementary metal–oxide–semiconductor (CMOS) based on
nuclear magnetic resonance (NMR) technique. The global market for in vitro diag-
nosis is expanding in both developed and developing countries ascribed to the grow-
ing population and longer life expectancy. Conventional benchtop tools for disease
diagnosis such as PCR (DNA amplification) are costly, bulky, and time-consuming
and require trained technicians for operation, which confound their usages in the
centralized laboratory.
CMOS is a promising alternative solution for rapid and quantitative diagnosis at
a low cost. It overcomes the miniaturization of healthcare diagnostic tools, allowing
low-cost and rapid detection of specific targets in tiny fluid samples. Among numer-
ous possible solutions to POC sensing mechanism, NMR stands out as a trailblazing
option since it is versatile and low-cost as it requires little processing on both the
samples and interfacing hardware, i.e., the transducers. However, the reported NMR
systems in literature encounter some issues such as bulky hardware, sample man-
agements, and magnetic field shifting. So herein the materials presented in this book
are focused on optimizing CMOS NMR platform for enhancing their applicability
by bridging NMR, semiconductor chips, and microfluidic technique and promoting
the application of NMR outside standard centralized laboratory with the aid of
CMOS chips. The proposed miniaturized NMR systems in this project achieve (1)
accurate and sensitive chemical/biological detection from microliter samples by the
CMOS integrated circuits; (2) electronic-automated sample management scheme
inside the space-limiting portable magnet, which significantly reduces the labors
and turnaround time of the assay; and (3) robust operation against environmental

v
vi Preface

variation such as temperature or displacement of the sample. The platforms show

promise as robust and portable diagnostic devices for a wide variety of biological
analyses and screening applications.
We hope the readers will enjoy the contents of this book.

Macao, China Ka-Meng Lei

July 2017
Pui-In Mak
Man-Kay Law
Rui Paulo Martins
Contents

1 Introduction.............................................................................................  1
1.1 Overview ......................................................................................... 1
1.2 Global Necessities for In Vitro Diagnostic Tools............................  2
1.3 Nuclear Magnetic Resonance for In Vitro Diagnosis......................  4
1.4 Organization.....................................................................................  6
References................................................................................................. 7
2 State-of-the-Art CMOS In Vitro Diagnostic Devices........................... 11
2.1 Introduction...................................................................................... 11
2.2 Transducing Mechanisms of CMOS IVD Tools.............................. 11
2.2.1 Electrical-Based................................................................... 12
2.2.2 Optical-Based....................................................................... 18
2.2.3 Magnetic-Based................................................................... 20
2.2.4 Mechanical-Based................................................................ 21
2.2.5 NMR-Based......................................................................... 25
2.3 In Vitro Diagnostic Applications...................................................... 26
2.3.1 Immunoassay....................................................................... 26
2.3.2 DNA Hybridization Assay................................................... 28
2.3.3 Cell/Bacteria Diagnosis....................................................... 29
2.4 Discussions and Selection Guide..................................................... 32
2.4.1 Integration Level.................................................................. 32
2.4.2 Labeling ............................................................................. 32
2.4.3 Hardware Preparation.......................................................... 33
2.4.4 Operation............................................................................. 33
2.4.5 Specificity............................................................................ 34
2.4.6 Summary ............................................................................. 34
References................................................................................................. 36

vii
viii Contents

3 Electronic-Automated Micro-NMR Assay with DMF Device............. 41

3.1 Introduction...................................................................................... 41
3.2 First Prototype: Primary Investigation on NMR–DMF................... 42
3.2.1 Discrete Electronics and Back-End Signal Processing........ 43
3.2.2 Magnet ............................................................................... 44
3.2.3 RF Coils ............................................................................. 44
3.2.4 DMF Device Fabrication and Actuation.............................. 46
3.2.5 Experimental Results........................................................... 47
3.3 Second Prototype: CMOS Micro-NMR Platform with DMF.......... 51
3.3.1 Design and Implementation of CMOS TRX....................... 53
3.3.2 Portable Magnet and RF Coil Codesign.............................. 58
3.3.3 DMF Device and Its Control Circuit................................... 60
3.3.4 Experimental Results........................................................... 60
3.3.5 Discussion and Outlook....................................................... 67
3.4 Summary ......................................................................................... 68
References.................................................................................................  69
4 One-Chip Micro-NMR Platform with B0-Field Stabilization............. 73
4.1 Introduction...................................................................................... 73
4.2 Platform Design............................................................................... 74
4.2.1 Micro-NMR Transceiver...................................................... 75
4.2.2 Multifunctional Planar Coil................................................. 76
4.2.3 Hall Sensor, Readout Circuit, and Current Driver............... 76
4.3 Prototype and Experimental Results................................................ 82
4.3.1 Experimental Setup and Electrical Measurements.............. 82
4.3.2 Biological/Chemical Measurements.................................... 84
4.3.3 Comparison and Discussion................................................. 86
4.4 Summary ......................................................................................... 88
References.................................................................................................  89
5 Conclusion and Outlook......................................................................... 91
5.1 Summary of Researches................................................................... 91
5.2 Future Prospects............................................................................... 92
References.................................................................................................  92

Appendix A: Modular NMR Electronic Components and

Measurement................................................................................................... 95

Appendix B: DMF Device and Electronics................................................... 97

Appendix C: Software and Hardware Interface of

Micro-NMR Platform..................................................................................... 99

Index................................................................................................................. 101
List of Abbreviations

1/f noise Flicker noise

AC Alternating current
ADC Analog-to-digital converter
AIDS Acquired immune deficiency syndrome
B0-field Static magnetic field
B1-field Radio-frequency magnetic field
BJT Bipolar junction transistor
BW Bandwidth
CMOS Complementary metal–oxide–semiconductor
CPMG Carr–Purcell–Meiboom–Gill
DAC Digital-to-analog converter
DC Direct current
DMF Digital microfluidic
DNA Deoxyribonucleic acid
EC Eddy current
EIS Electrochemical impedance spectroscopy
ELISA Enzyme-linked immunosorbent assay
EWOD Electrowetting-on-dielectric
FoM Figure of merit
FPGA Field-programmable gate array
GBW Gain–bandwidth product
hCG Human chorionic gonadotropin
HIV Human immunodeficiency virus
hMAM Human mammaglobin
IC Integrated circuit
IDT Interdigital transducer
IF Intermediate frequency
IgG Immunoglobulin G
IgY Immunoglobulin Y
IIP3 Third-order intercept point
IRN Input-referred noise

ix
x List of Abbreviations

ISFET Ion-sensitive field-effect transistor

ITO Indium tin oxide
IVD In vitro diagnostic
LNA Low-noise amplifier
LO Local oscillator
LOC Lab-on-a-chip
LoD Limit of detection
LPF Low-pass filter
LSB Least significant bit
MNP Magnetic nanoparticle
MOSFET Metal–oxide–semiconductor field-effect transistor
MP Magnetic particle
MUX Multiplexer
NMOS N-channel MOSFET
NMR Nuclear magnetic resonance
NW Nanowire
PA Power amplifier
PBS Phosphate-buffered saline
PC Personal computer
PCB Printed circuit board
PLL Phase-locked loop
PM Phase margin
PMOS P-channel MOSFET
PNIPAM Poly(N-isopropylacrylamide)
PoC Point of care
PoU Point of use
PSS Pulse sequence synthesizer
qPCR Quantitative polymerase chain reaction
RBC Red blood cell
RF Radio-frequency
RX Receiver
SAL Supercritical angle luminescence
SAW Surface acoustic wave
SNR Signal-to-noise ratio
SPAD Single-photon avalanche diode
TAT Turnaround time
THD Total harmonic distortion
TIA Transimpedance amplifier
TRX Transceiver
TX Transmitter
UART Universal asynchronous receiver/transmitter
VHS Vertical Hall sensor
WHO World Health Organization
XO Crystal oscillator
β-LG β-Lactoglobulin
List of Figures

Fig. 1.1 World population from 1950 to 2050, with a medium variant
estimation from 2015. Data collected from the United Nations
World Population Prospects: The 2015 Revision [14].
More developed countries: countries in Europe and Northern America,
plus Australia/New Zealand and Japan. Less developed countries:
countries in Africa, Asia (except Japan), Latin America, and the
Caribbean plus Melanesia, Micronesia, and Polynesia................. 2
Fig. 1.2 The old age dependency ratio (solid line), which is defined
as the ratio of population of 65+ years old to the population
of 15–64 years old with medium variant estimation from 2015.
The children dependency ratio (dotted line), which is defined as the
ratio of population of 0–14 years old to the population of 15–64 years
old, is also shown on the graph as reference. Data collected from the
United Nations World Population Prospects: The 2015 Revision [14].
More developed countries: countries in Europe and Northern America
plus Australia/New Zealand and Japan. Less developed countries:
countries in Africa, Asia (except Japan), Latin America, and the
Caribbean plus Melanesia, Micronesia, and Polynesia................. 3
Fig. 1.3 (a) Macroscopic view of the non-zero spin nuclei. With an external
magnetic field B0 applied to the nuclei, part of them will align
with this magnetic field. (b) The effect of RF excitation
on the nucleus under external magnetization. When excited
by the RF magnetic field at fL, the nuclei precess around the
magnetization. After this excitation, the nuclei still resonate
and return to the equilibrium, with this relaxation recorded
and analyzed.................................................................................. 4
Fig. 1.4 The state of the probe-functionalized MNPs. (a) Without the target,
the MNPs stay monodisperse in the solution without
any aggregation. (b) When the targets exist in the sample,
the targets bind with the probe, and the MNPs aggregate
to form micro-clusters.................................................................... 6

xi
xii List of Figures

Fig. 2.1 Architecture and operation of electrical-based detection CMOS

biosensor. An extra layer of noble and biocompatible metal
such as gold is deposited on the original built-in metal layer.
The capturing probe is then immobilized on the gold electrode
to capture the target. Upon hybridization the electrical properties
such as impedance or charge are sensed directly by the
readout circuit................................................................................ 16
Fig. 2.2 Cell culturing and monitoring with CMOS capacitive sensing chip.
(a) The photograph showing the overall chip with dual in-line
package. A well encloses the cell culturing site, and the CMOS
chip is at the center of the well. The polymer protects the bond
wires of the chip. (b) Photomicrograph of the electrodes.
Since the system measures only the capacitance of the single
electrode, the built-in passivation layer such as silicon nitride and
silicon dioxide can be preserved without further post-processing.
This simplifies the hardware preparation steps for biosensing.
(c) The experimental results for cancer cell MDA-MB-231 culturing.
The capacitance at specific site increases
due to the proliferation of the cancer cells ascribed to the increased
number of cells, allowing real-time monitoring for the growth
of the cancer cells (Reproduced with permission from [38].
Copyright 2008 Elsevier)............................................................... 17
Fig. 2.3 Architecture and operation of optical-based detection CMOS
biosensor. The capturing probe is immobilized on a solid substrate
such as glass or the built-in passivation layer atop the CMOS chip.
Then fluorescence-labeled or chemiluminescence-labeled target
will bind with the probe, and other unbound biomolecules will be
washed away. The CMOS photodetector, which is formed by the
embedded PN-junction, transduces the optical signal to current for
subsequent signal processing......................................................... 18
Fig. 2.4 Lens-free cell/microparticle counting system with CMOS
image sensor. (a) The overall platform of the digital cell counting
device. (b) The micrograph of the microcavity array for cell trapping.
The sample under analysis is put atop the microcavity array.
Then the suspended cells/microparticles will be pulled toward
and trapped in the cavities attributed to the negative pressure.
This negative pressure is produced by peristaltic pump, which
extracts the air inside the chamber. (c) Detection principle of the
system. The light from the external UV light source will arrive
at the CMOS image sensor through the unoccupied cavity,
while the trapped cell on the cavity blocks the light from arriving
at the CMOS image sensor. (d) The schematics of the expected
CMOS image acquired from (c). Since the cell blocks the UV light
from passing through the cavity, the pixels under those occupied
cavity will report a darker region, while the pixels under the vacant
List of Figures xiii

cavity will report a brighter result. Thus the number of cells

on the microcavity array can be identified from the result
of the CMOS image sensor (Reproduced with permission from [43].
Copyright 2014 Saeki et al.).......................................................... 20
Fig. 2.5 Architecture and operation of magnetic-based detection CMOS
biosensor. The capturing probe is immobilized on a solid substrate
such as glass or the built-in passivation layer atop the CMOS chip.
Then the sample labeled with MP will mix with the capturing probe.
Matched target will be captured, and unbound objects will then be
rinsed off. A magnetic transducer such as LC oscillator or Hall
sensor will transduce the magnetism of the sample to electrical
signals, which will be processed by the readout circuit
subsequently.................................................................................. 21
Fig. 2.6 The magnetic-based handheld diagnostic device for antigen
and nucleic acid detection. (a) The overall diagnostic device.
The CMOS chip can be easily connected with the PCB by a cartridge.
(b) The disposable cartridge with the CMOS chip. The CMOS chip
is attached to the cartridge with silver epoxy and connected with
bond wires to the carrier leads. This arrangement enables a disposable,
low-cost, and multiplexed assay and simplifies the sample handling
module such as microfluidic to manage the sample to the sensing
sites. (c) The CMOS chip. It has 48 on-chip sensing sites together
with 16 reference sensors. Each coil together with its own capacitor
forms an LC oscillator, which has an oscillating frequency inversely
proportional to the square root of the inductance of the coil.
The surface of the chip is bio-functionalized for probe immobilization.
The sample with the MP is then applied to the surface of the chip,
followed by a washing step to rinse the unbound molecules and MPs.
The bound MPs increase the inductance of the coils. Thus by detecting
the oscillation frequency, the concentration of the target at the specific
site can be selectively evaluated. (d) The experimental results for
DNA detection. The frequency shift of the oscillation frequency is
commensurate with the concentration of the target. With the novel
magnetic freezing scheme, a limit of detection of 100 pM DNA
can be achieved (Reproduced with permission from [22]. Published
by the RSC 2014)........................................................................... 22
Fig. 2.7 Architecture and operation of mechanical-based detection CMOS
biosensor. (a) Mechanical-based detection with cantilever.
A cantilever can be exploited to transduce the mass attached on
it to electrical signals such as resistance. A gold layer is deposited
on the cantilever for growing the capturing probe on it. In order
to allow the cantilever to bend upon the biomolecule attached,
the neighbor insulating dielectrics and the base of the cantilever
are etched away. A piezoresistor can be adopted to transduce the
bending force on the cantilever to resistance change, and the readout
xiv List of Figures

circuit will detect this variation. (b) Mechanical-based detection

with SAW transducer. A complete SAW transducer consists of three
modules, input metal interdigital transducer (IDT), the piezoelectric
delay line where the acoustic wave travels through, and the output
metal IDT. The input IDT generates the SAW. Then the wave travels
through the delay line to the output IDT, where the SAW is transduced
back to the electrical signal. The bio-functionalized gold layer atop
the delay line captures the entity under analysis. The increased
mass here will affect the characteristics of the delay line, resulting
in change of resonant frequency, amplitude, or phase shift
on the SAW, which then can be detected on the output IDT......... 23
Fig. 2.8 A CMOS cantilever-based biosensor for DNA detection.
(a) The operation procedures of the biosensor. After post-processing
to implement the cantilever on the CMOS chip, the capturing DNA
is then immobilized on the Au surface of the cantilever. Then the
cantilever is immersed in the PBS buffer, and the sample of interest is
injected around the cantilever to allow hybridization of DNA. After
washing unbound biomolecule, the cantilever is left to dry. After all of
the water molecules are evaporated, the matched target DNA will stay
on the Au surface. Their masses incur bending of the cantilever,
and an embedded piezoresistor implemented by N+ polysilicon
is entailed to sense this bending and transduce it to variation of
its own resistance, causing a frequency shift on the ring-type
oscillator. (b) The SEM image of the cantilevers. In order to allow
the cantilever bending freely in air, the surrounding materials
such as the insulating dielectrics and underneath the p-substrate have
to be etched away, creating a suspending cantilever. (c) Experimental
results for the biosensor. The resistance variation of the polysilicon
piezoresistor attributed to the bending of the cantilever incurs in a
deviation of the oscillating frequency. After DNA sample injection,
washing, and drying steps, the final steady-state frequency can be
measured to selectively quantify the concentration of the target
DNA inside the sample with limit of detection of 1 pM
from hepatitis B virus (Reproduced with permission from [19].
Copyright 2013 IEEE)................................................................... 24
Fig. 2.9 Architecture and operation of NMR-based detection CMOS
biosensor. NMR focuses on the measurement of the NMR signals
from the samples. First, the MNP functionalized with the capture
probes reacts with the sample under analysis. Then the mixture
will be put atop the spiral sensing coil to perform NMR
experiment. The existence of target inside the sample incurs
in MNPs aggregation; thus a larger micro-cluster will
be formed, changing the spin–spin relaxation time
of the NMR signal from the sample.............................................. 25
List of Figures xv

Fig. 2.10 The one-chip CMOS NMR-based biosensor. (a) The prototype
of the platform. The system consists of a portable permanent
magnet for magnetizing the 1H nuclei and the CMOS chip to excite
the nuclei and receive the NMR signal from them. The samples
are put directly on top of the CMOS chip without further
post-processing. (b) The experimental results from the biological
samples. Without the target the functionalized MNPs stay
monodispersed, and the sample has a higher T2. With the target
hCG cancer marker inside the samples, the hCG antibody binds
with the hCG cancer marker, and they together form the
micro-cluster. Thus the T2 of the sample decreased, and the
concentration of the target can be identified from the NMR
signal (Reproduced with permission from [28].
Copyright 2011 IEEE)................................................................... 27
Fig. 2.11 Smart CMOS system-on-chip platform for rapid blood screening
test of risk prediction. (a) The experimental procedure of the platform.
Firstly, the blood under analysis is put atop the anodic aluminum
oxide membrane. The biomarkers will be diffused to the mixing
reservoir and separated from other blood cells (>1 μm). After the
filtration, the filtered sample in the mixing reservoir together with
the bio-functionalized magnetic bead will be pumped to the sensing
site by the force from the electrolytic pumping. Upon capturing
by the coated antibody at the surface of the CMOS chips, the target
and the magnetic bead will be seized, while the unbound magnetic
bead will be flushed away by the magnetic force from the on-chip
coil. Thus the Hall sensor can sense the magnetic bead and identify
the concentration of the targeted biomarker. (b) The photograph
showing the electrolytic pumping and magnetic flushing. At first,
the sample is on the right of the sensing reservoir. Then, voltage
is applied to the electrolytic electrodes, and bubbles are formed
consequently. The bubbles here induce gas force and pump the
sample to the sensing reservoir. After the sample arrived at the
sensing site, the immobilized antibodies capture the targets
and the magnetic beads. Then the unbound magnetic beads
will be flushed away by the on-chip coil. (c) The experimental
result (TNF-alpha) of the immunoassay. The Hall sensor detects
the target analyte from the magnetic beads on the sensing site.
The system can detect 0.8 pg/mL–80 ng/mL of TNF-alpha
and NT-proBNP from whole bloods (Reproduced with
permission from [34]. Copyright 2015 IEEE)............................... 28
Fig. 2.12 Integrated qPCR system on CMOS chip. (a) The CMOS chip
and illustration of its functions. The chip has three main modules
to enable on-chip qPCR. An electrowetting-on-dielectric device
serves as an electronic-automated droplet management module
to extract the target, PCR reagents, buffer, and intercalator dye
xvi List of Figures

from the reservoirs, respectively, and guides them to different

electrodes for mixing and subsequent operations by applying voltage
on corresponding electrodes. A thermal module, which includes a
resistive heater and temperature sensor, regulates the temperature
of the droplets to perform thermal cycling for PCR. SPAD is embodied
on the CMOS chip to detect the fluorescent emission from the target
DNA in real time for qPCR. (b) Experimental results of the qPCR.
The fluorescent signal from the sample increases with the PCR cycle.
The qPCR system achieves a linear relationship between the cycle
threshold and logarithm of initial DNA concentration from 1 to 10,000
copies per 1.2 nL of droplet, resulting in a 40,000-fold of reduction
on reagent consumption (Reproduced with permission from [24].
Copyright 2014 RSC publishing)................................................... 30
Fig. 2.13 CMOS multimodal sensor array for cell-based assay. (a) Schematic
of the multimodal cell-based assay platform. The entire platform
consists of 3 × 3 sensor array, and each pixel consisted
of a photodiode, a temperature sensor (shared within a pixel group),
a voltage amplifier, and an impedance detector for multimodal study
and monitoring of the cultured cell exposed to drug or pathogen
stimulation. (b) The micrograph of the CMOS cellular sensor chip.
The chip contains 9 pixel groups for individual cell-based assay,
and each pixel group further contains 16 individual pixels. Each pixel
is formed by a gold-plated electrode for action potential and
impedance reading with a photodiode. (c) Real-time experimental
results from the bioluminescence experiment at 2 pixels. The human
ovarian cancer cell emits luminescence upon the addition of luciferin,
enabling verification of cell viability. The photodiode captures this
bioluminescence, and the readout circuit processes the signal for
subsequent analysis (Reproduced with permission from [48].
Copyright 2015 IEEE)................................................................... 31
Fig. 2.14 A radar chart showing the conceptual requisites to perform the
in vitro diagnosis on biomolecule targeting with different
transducing mechanism.................................................................. 35
Fig. 3.1 The overall schematic and operations of the NMR–DMF system.
(a) The placement of the DMF device, magnet, RF coil, and PCB
in 3D view; (b) schematic of the NMR electronics; (c) the filtered
results from the PCB are captured by the oscilloscope for easier
demonstration purpose and then analyzed in MATLAB; (d) the
photograph of the DMF device and its structure; (e) the detection
mechanism of the NMR–DMF system. The target-specific
MNPs, which act as probes, are placed on the sensing site initially
(in purple). The sample at other electrodes (in cyan)
will be transported to the sensing site and mixed with the probe
to perform NMR assay................................................................... 42
List of Figures xvii

Fig. 3.2 Timing diagram of the pulses, including the excitation CPMG
pulse sequence delivered to the TX to excite the nuclei and the
response from the nuclei, which is picked up by the coil.............. 43
Fig. 3.3 (a) Geometry and limitation from the opening gap of the portable
magnet. (b, c) The EM simulation of the magnetic field direction
and strength from a spiral coil (with 14 turns) and a Butterfly-coil
(with 7 turns on each spiral), respectively, with a flowing
current of 1 A................................................................................. 45
Fig. 3.4 Ratio of EC loss generated by the seven-turn (each loop)
Butterfly-coil to coil magnetic energy against the thickness
of the ITO. The figure was plotted based on (3.2) with f = 20 MHz,
ρ = 1 × 10−6 Ωm, and A = 40 mm × 24 mm. The dotted line shows
0.5% level and corresponds to the ITO thickness of 80 nm.......... 47
Fig. 3.5 Nutation curve of the seven-turn (each loop) Butterfly-coil. The
normalized amplitude from different durations of RF excitation
signals was recorded and fitted to the sinusoidal wave.
The estimated π/2-pulse width for the coil is 144 μs..................... 49
Fig. 3.6 (a) Received NMR signal from water. Inset shows the received
NMR signal. The echoes were bounded by the gray-dotted trend line.
(b) T2 of the samples versus concentration of CuSO4 solution, and
results were shown on the graph (■). The trend lines were drawn
together with their equation and 1/T2 value, together with error
percentages (defined as half of 95% confidence level/true value)
marked on the graph with dot lines where the values
were displayed on the right axis.................................................... 50
Fig. 3.7 (a) Fabricated DMF device. For illustration, the electrodes
are numbered 1–8; (b, c) operation of the DMF platform.
The droplet was originally placed at electrode no. 1
(highlighted by the circle). By applying a signal on electrode
no. 2 and then turning off electrode no. 1, the droplet moved
to electrode no. 2. As such, the droplet can be transported
to electrode no. 8, which is the NMR sensing site......................... 51
Fig. 3.8 (a) Illustration of droplets mixing. The droplets at electrode no. 1
(samples) and no. 8 (probe) were driven to electrode no. 7
and mixed together. (b) The NMR assay results
from the mixed droplets................................................................. 52
Fig. 3.9 Portable electronic-automated micro-NMR system. It features
a CMOS TRX and a PCB-based Butterfly-coil inside the magnet
to transduce between magnetic and voltage signals. The analyte
is placed inside a glass substrate DMF device atop the
Butterfly-coil for sample management (only one electrode
is shown for simplicity)................................................................. 52
Fig. 3.10 Three-phase operation of the micro-NMR system: setup,
sample preparation, and analysis................................................... 53
xviii List of Figures

Fig. 3.11 Block diagram of the micro-NMR TRX cooperated with

the DMF device. It includes a CMOS micro-NMR TRX
with a Butterfly-coil input, a DMF device, and DMF electronic.
An electrode has the Butterfly-coil placed underneath
for performing micro-NMR assays. An FPGA connected
to a computer coordinates the hardware........................................ 54
Fig. 3.12 Pulse-sequence synthesizer. FPGA commands control the logic
gates to master the start and duration of the excitation signals
with different phases as well as the switching between TX
and RX modes................................................................................ 55
Fig. 3.13 (a) Butterfly-coil-input LNA and its noise model.
(b) Double-balance quadrature mixer with RF-sharing stage.
(c) Source-follower-based tunable bandwidth LPF....................... 56
Fig. 3.14 Simulation results of the mixer with LO = 20 MHz and input
frequency = 20.002 MHz (i.e., IF = 2 kHz): (a) output against
input for fundamental and third harmonic. (b) THD of the mixer
at different input amplitudes.......................................................... 57
Fig. 3.15 (a) Simulated pole plot of the LPF. The sixth-order LPF
implements a Butterworth filter (poles form a semicircle) with
various cutoff frequencies obtained by changing only their bias
currents. (b) Simulated THD of the LPF with an input frequency
of 2 kHz and a cutoff frequency of 5 kHz for different
input levels..................................................................................... 58
Fig. 3.16 The micro-NMR pulse sequence. It includes the CPMG pulse,
filter current control, and micro-NMR output signal
where the dead time of the RX is shown....................................... 59
Fig. 3.17 Simulated SNR of the Butterfly-coil-input CMOS RX
with different number of turns in the coils.................................... 60
Fig. 3.18 (a) Chip photo. (b) Measured performance summary
of the micro-NMR TRX. The RX’s IRN, gain, and BW can only
be assessed by simulations as the RX input has been tied
to the Butterfly-coil........................................................................ 61
Fig. 3.19 (a) Block diagram of the image-reject RX. (b) Measured RX output
spectrum with an externally coupled magnetic field at 19.999 MHz
and a LO of 20 MHz after image noise removal. (c) Cutoff frequency
and settling time of the LPF versus the bias current.
Working regions of the LPF at different modes are labeled.......... 62
Fig. 3.20 Measured B0 with and without calibration..................................... 63
Fig. 3.21 The system hardware of the micro-NMR system. It is linked
with an FPGA (DE0-Nano) and a program implemented in C# which
facilitates the system control, result collection, and displays........ 63
Fig. 3.22 The pulses counted on the electrodes covered by air and water,
respectively. As the permittivity of water is substantially larger
than air (80:1), the capacitance of the electrode covered by water
is higher, causing lower pulses to be counted, and thus the system
can detect if the electrode is vacant............................................... 64
List of Figures xix

Fig. 3.23 Operation of the micro-NMR system. (a) Initial position

of the sample and its projected path. (b) Droplet moves to the
adjacent electrode. (c) Final position (micro-NMR sensing site)
of the droplet. (d) Measured micro-NMR signal from water droplet
excited by CPMG pulse sequence with 256 echoes and 4 ms interval.
The envelope is extracted and fitted to a mono-exponential function,
as shown in the inset...................................................................... 65
Fig. 3.24 (a) The correlation of ΔT2−1 (with reference to 0 mM of CuSO4)
with the concentration of CuSO4. The echoes amplitude for the case
of CuSO4 at 1 mM concentration is plotted above. One hundred
twenty-eight echoes were collected for each single experiment.
(b) The correlation of ΔT2 (with reference to 0 μM of avidin)
with the concentration of avidin. The echoes amplitude for the case
of avidin at 0.2 μM concentration is plotted above. Sixty-four echoes
are collected for each single experiment....................................... 66
Fig. 3.25 (a) Illustration of the motions of the droplets for multistep
multi-sample handling. T2 for the water sample: 256 ms; for avidin:
211 ms. (b) A Gantt chart of the operation of an individual droplet.
The total time for the experiment is 2.2 min................................. 67
Fig. 4.1 Conceptual diagram of the proposed micro-NMR platform for PoU
applications. Different samples such as protein and DNA can be
put directly atop the CMOS chip for assays. A portable magnet
is entailed to magnetize the nuclei inside the samples.................. 74
Fig. 4.2 System block diagram. The TX and RX transduce between
magnetic and electrical signals with a thermal-controlled spiral coil.
The B0-field sensor and calibrator automatically stabilize the bulk
magnetization on the μL sample. No frequency synthesizer
is required...................................................................................... 75
Fig. 4.3 (a) Simulated 3D temperature distribution of the droplet at applied
power of 8 mW in COMSOL Multiphysics®; (b) Simulated droplet
average temperature at applied power from 0 to 20 mW............... 76
Fig. 4.4 The cross section of a single VHS element and its current path.
(a) Without lateral magnetic field; (b) with lateral magnetic
field B0........................................................................................... 77
Fig. 4.5 Proposed current-mode fourfolded VHS arranged in Wheatstone
bridge to sense the lateral B0-field and its readout circuit
(spinning circuitry is omitted for simplicity). The latter features
a nominal B0-field compensator to offset the strong nominal
B0-field (0.46 T) for better sensitivity (3.75 mT). The green arrows
highlight the current paths of IHall. Inset shows the timing diagram
for the switches and overall operations.......................................... 78
Fig. 4.6 Illustration for the two-phase spinning technique on the VHS.
The bias direction (U1 and U3) together with the output terminals
(U2 and U4) of the VHS is swapped periodically to eliminate
the 1/f noise and offset of the elements.......................................... 78
Fig. 4.7 Simulated frequency response of the TIA with various TINT......... 79
xx List of Figures

Fig. 4.8 Simulated channel resistance (RDS) and parasitic capacitance

(CS+CD) of the MOS versus channel width................................... 80
Fig. 4.9 Simulated output waveforms of the integrator. Without the nominal
B0-field compensator, the integrator is saturated due to the large
current induced by the nominal B0-field before it accumulates
an adequate voltage difference. Whereas with the compensator,
the nominal B0-field can be compensated; thus, the integration
time can be prolonged to produce sufficient voltage differences
at the output................................................................................... 81
Fig. 4.10 (a) Chip photo of the fabricated chip in 0.18-μm CMOS.
(b) Prototype of the micro-NMR platform with B0-field stabilization
and lab-on-a-chip feasibility for multi-type biological/chemical
assays, including (1) permanent magnet, (2) CMOS micro-NMR
chip (inside magnet), (3) PCB, (4) FPGA, and (5) current driver.
(c) Experimental setup. A program developed in C# is entailed
for hardware control and visualizing the experimental results.
The platform is powered by two batteries for portability.............. 82
Fig. 4.11 Timing diagram of the B0-field calibration and
its frequency-domain illustration................................................... 83
Fig. 4.12 (a) Measured hall sensor response; (b) B0-field with and without
calibration. Actual B0-field is the sum of the B0-fields
from the permanent magnet and the auxiliary coil driven
by the current driver....................................................................... 84
Fig. 4.13 (a) Measured power consumption and FoM of the XO
at different supply voltages. (b) Measured phase noise of the XO
(VDD = 0.9 V, f = 78.5 MHz). Compared with the LO generated
from signal generator (Agilent 3350A), the XO shows a much
better phase noise at low power..................................................... 84
Fig. 4.14 Experimental results from biological samples. (a) Target
quantification from human IgG as target and chicken IgY
as control. (b) Target quantification from Enterococcus faecalis-
derived DNA together with single-base mismatch DNA............... 85
Fig. 4.15 Experimental results from biological/chemical samples.
(a) Protein (β-LG) state detection with different heating temperature.
(b) Polymer (PNIPAM) dynamics with the solvent during heating
from the on-chip heater.................................................................. 86
Fig. A.1 Measured gain of the NMR RX..................................................... 96
Fig. A.2 Measured output spectrum of the RX with a 100-nV, 20-MHz
sinusoidal input.............................................................................. 96
Fig. B.1 Visualized waveform applied to the electrode before
and after the droplet arrives at the electrode.................................. 98
Fig. C.1 The communication between the PC and the FPGA board to drive
the micro-NMR relaxometer. It is done by adopting the TTL-232R_
PCB module to interfacing between the PC and FPGA board,
which mastered the hardware of the micro-NMR relaxometer..... 100
List of Tables

Table 2.1 Recent CMOS-based DNA-related biosensors............................. 13

Table 2.2 Recent CMOS-based protein-related biosensors........................... 14
Table 2.3 Recent CMOS-based cell-related biosensors................................ 15
Table 3.1 Summary of the measured and simulated coil parameters
at 20 MHz...................................................................................... 48
Table 3.2 Simulated noise summary of the LNA.......................................... 56
Table 3.3 Comparison with the existing CMOS-based NMR system........... 68
Table 4.1 Summary and benchmark with other CMOS-based
PoU systems.................................................................................. 87
Table 4.2 Benchmark with previous CMOS NMR systems.......................... 88

xxi
Chapter 1
Introduction

1.1 Overview

An essential part to evaluate the success of global health is the access to appropriate
diagnostic tools [1]. A commendable diagnostic tool should be able to identify the
disease occurred from the individuals rapidly. Especially for the infectious diseases,
the turnaround time (TAT) for the diagnosis strongly affects their exacerbation level
to the community. In vitro diagnostic (IVD) tool is aimed to offer a comfortable
diagnosis for the patients, by taking only small specimens from the human body,
e.g., blood, urine, or sputum, for analysis. Consequently, technologies enabling
effective in vitro diagnosis become highly attractive for both developed and devel-
oping countries [2]. Tremendous efforts have been geared toward developing
clinical-­level IVD tools. Despite achieving high accuracy, the resulting TAT can be
too long for diagnoses of contagious diseases like Ebola and SARS in the rural area,
and the requisite of skillful operators and sophisticated equipment to perform the
assays can dramatically raise the cost of the assay.
Recently, decentralized diagnostic solutions, namely, point-of-care (PoC)
devices, have gained notable interests attributed to their fastness, small footprint,
and tiny sample usage. Wide varieties of diagnostic platforms have been invented,
such as the lateral flow assays [3–6] and pathbreaking lab-on-a-disc immunoassay
[7–10] for PoC applications. Beyond them, PoC devices on complementary metal–
oxide–semiconductor (CMOS) chips are particularly promising, as they can enjoy
the maturity of microelectronics in manufacturing and its outstanding performances
in both physical sensing and signal processing. While the mainstream lateral flow
assay is confounded to provide merely qualitative or semiquantitative results [11],
the CMOS biosensors can attain a quantitative result and are beneficial to rapid and
low-cost assays. Especially for low-cost IVD applications, CMOS chips in a centi-
meter scale can significantly miniaturize the diagnostic tools.

© Springer International Publishing AG 2018 1

K.-M. Lei et al., Handheld Total Chemical and Biological Analysis Systems,
https://doi.org/10.1007/978-3-319-67825-2_1
2 1 Introduction

1.2 Global Necessities for In Vitro Diagnostic Tools

Decentralized healthcare systems are highly attractive for developing countries, as

they typically suffer from lack of access to high-quality centralized diagnostic tools
in the resource-limited area. Delay of diagnosis and treatment aggravates the health-
care condition of their countries, then affecting the global health system. According
to the World Development Report in 2004, the lack of access results in failure of the
health services [12]. Without proper equipment for diagnosis, the clinicians could
only diagnose diseases from the clinical symptoms in resource-limited regions. Yet,
this may cause difficulties in the diagnosis of the patients when the symptoms are
still unobvious. Especially for infectious diseases, the delay of treatment can worsen
the situation of individuals and consequently the communities. According to the
report of the World Health Organization (WHO), the leading infectious diseases
(lower respiratory infections, HIV/AIDS, diarrheal diseases, malaria, and tubercu-
losis) account for roughly one-third of all deaths in low-income countries [13].
Also, the strong growths of the population in those areas give rise to the demand of
affordable IVD tools. By the end of 2050, the less developed countries are expected
to have a population of 8.4 billion, as depicted in Fig. 1.1, where Africa and Asia
contribute roughly 2.48 and 5.27 billion, respectively [14]. Thus, there is a rapidly
growing market of low-cost PoC devices for developing countries.

More Developed Countries Less Developed Countries

More Developed Countries (Est.) Less Developed Countries (Est.)
12

10
Population (Billion)

0
1950 1960 1970 1980 1990 2000 2010 2020 2030 2040 2050
Year

Fig. 1.1 World population from 1950 to 2050, with a medium variant estimation from 2015. Data
collected from the United Nations World Population Prospects: The 2015 Revision [14]. More
developed countries: countries in Europe and Northern America, plus Australia/New Zealand and
Japan. Less developed countries: countries in Africa, Asia (except Japan), Latin America, and the
Caribbean plus Melanesia, Micronesia, and Polynesia
1.2 Global Necessities for In Vitro Diagnostic Tools 3

More Developed Countries Less Developed Countries

More Developed Countries (Est.) Less Developed Countries (Est.)
0.9

0.8

0.7

0.6

0.5
Ratio

0.4

0.3

0.2

0.1

0
1950 1960 1970 1980 1990 2000 2010 2020 2030 2040 2050
Year

Fig. 1.2 The old age dependency ratio (solid line), which is defined as the ratio of population of
65+ years old to the population of 15–64 years old with medium variant estimation from 2015. The
children dependency ratio (dotted line), which is defined as the ratio of population of 0–14 years
old to the population of 15–64 years old, is also shown on the graph as reference. Data collected
from the United Nations World Population Prospects: The 2015 Revision [14]. More developed
countries: countries in Europe and Northern America plus Australia/New Zealand and Japan. Less
developed countries: countries in Africa, Asia (except Japan), Latin America, and the Caribbean
plus Melanesia, Micronesia, and Polynesia

The aging problem of the developed countries also creates an enormous chal-
lenge. A healthcare solution that can deal with the continuous increment of life
longevity is of demand. As revealed in Fig. 1.2, the old age dependency ratio, which
gives insight to the population of elderly (65+ years) as a share of those in working
age (age 15–64 years), will be rising in the coming decades. The old age depen-
dency ratio of more developed countries will reach 0.4 in 2034 and eventually 0.46
by the end of 2050 (i.e., increase by 72% from 2015). Thus, the burdens on the clini-
cal resources in those areas will become tighter, especially for the patients in prox-
imity to death [15]. An efficacious healthcare solution will benefit this situation and
drive the growth of the market for IVD tools. To this end, the market for IVD tools
should not be merely aimed at less developing countries but also toward efficient
and convenient diagnosis in developed countries. In fact, according to the report
from Forbes/Investing, the IVD market, valued at ~$50 billion in 2012, will expand
to $70 billion by 2017 [16].
4 1 Introduction

1.3 Nuclear Magnetic Resonance for In Vitro Diagnosis

Nuclear magnetic resonance (NMR) is powerful to explore the sample information

at the molecular level. The underpinning physics of NMR is the exchange of energy
between the RF magnetic field and the spin of the non-zero spin nuclei (i.e., 1H, 13C,
17
O, 31P, etc.) [17, 18]. Under the magnetization with an external magnetic field B0,
parts of the nuclei align with this external magnetic field and have a spin-up state,
while the others have a spin-down state and align in opposite direction (Fig. 1.3a).
As the population ratio between the nuclei with spin-up and spin-down state is pro-
portional to B0 and this difference determines the amplitude of the NMR signal,
there exists a tradeoff between the portability and sensitivity of the system as dis-
cussed later. With an RF magnetic field B1 orthogonal to B0 applied to the nuclei,
they precess and tip away from the direction of bulk magnetization (Fig. 1.3b). The
nuclei only accept RF excitation at Larmor frequency, defined as:

fL = γ B0 (1.1)

Spin-up
Spin-down

Nuclei

Magnetic
moment
B0
Without external magnetization With external magnetization

(a)

B0
B1 (at f L)
Before RF excitation Right after RF excitation After RF excitation (relaxation)

(b)
Fig. 1.3 (a) Macroscopic view of the non-zero spin nuclei. With an external magnetic field B0
applied to the nuclei, part of them will align with this magnetic field. (b) The effect of RF excita-
tion on the nucleus under external magnetization. When excited by the RF magnetic field at fL, the
nuclei precess around the magnetization. After this excitation, the nuclei still resonate and return
to the equilibrium, with this relaxation recorded and analyzed
1.3 Nuclear Magnetic Resonance for In Vitro Diagnosis 5

with the gyromagnetic ratio of the nuclei γ. For a 0.46-T magnet, the fL of 1H is
~20 MHz. The nuclei do not precess if there is a mismatch on the excitation fre-
quency and fL. After tipping the nuclei with 90° from the direction of bulk magneti-
zation, the excitation is turned off. Then, the recovery of the magnetization in
parallel with B0 is defined as the spin-lattice relaxation time T1 whereas the recovery
of the magnetization perpendicular with B0 is defined as the spin-spin relaxation
time T2. This T2 reveals the magnetic field decoherence information across the
nuclei. Unfortunately, the unavoidable inhomogeneity of B0 from portable magnet
causes spatial variation on the precession rate of the nuclei thus the T2 decays at a
much faster rate T2*. This blemish hinders the measurement on original T2. To sur-
mount this, the spin-echo technique such as Carr–Purcell–Meiboom–Gill (CPMG)
pulse sequence can be utilized to refocus this dephasing effect on the nuclei by flip-
ping the nuclei 180° with interval τ; thus the spins are maximized again from B0
inhomogeneity [19, 20]. The envelope of the echoes responses allows the derivation
of the resulting T2 with the following mathematical expression:

nτ
−
A [ n ] = A0 e T2
(1.2)

with the echoes amplitude for nth echoes A[n] and the initial amplitude A0. More
importantly, the strength of B0 correlates to the signal-to-noise ratio (SNR) of the
NMR signal and is described as [21]:
7
 fL  4
1  2π 
SNR NMR ∝ KB1 (1.3)
Flξ∆ f 4 ρ

with homogeneity factor K, magnetic field strength per unit current produced by the
RF-coil orthogonal to the permanent magnetic field B1, the noise figure of the
receiver’s forefront amplifier F, length of the RF-coil conductor l, the bandwidth of
the system Δf, and the resistivity ρ of the RF coil. From (1.1) and (1.3), the SNR of
the system is proportional to the power of 7/4 of B0, thus demanding a stronger B0
to enhance the SNR. Although there seem to be numerous ways to enhance B0 and
its homogeneity (i.e., for higher resolution and stronger signal), the portability and
power consumption of the system will be penalized due to the need for a heavier and
bulkier magnet, not to mention a higher operating frequency that will be required
for the electronics.
By exploiting functionalized magnetic nanoparticles (MNPs) as the probe, the
NMR-based quasi label-free detection scheme can pinpoint a broad range of unpro-
cessed biological targets such as DNA [22], protein [23], and virus [24] for in vitro
diagnosis. These superparamagnetic MNPs have significant impacts on T2 of the
samples according to their magnetization (Ms) attributed to their capability to per-
turb the local magnetic field homogeneity. When the target is absent in the sample,
the MNPs stay monodisperse inside the solution (Fig. 1.4a). Consequently, when
6 1 Introduction

MNP

Probe

Target
(a) (b)

Fig. 1.4 The state of the probe-functionalized MNPs. (a) Without the target, the MNPs stay
monodisperse in the solution without any aggregation. (b) When the targets exist in the sample, the
targets bind with the probe, and the MNPs aggregate to form micro-clusters

the targets exist inside the samples, they will cross-link with the probe-­functionalized
MNPs, assembling nanoparticles micro-clusters (Fig. 1.4b). These micro-clusters,
with a diameter dc depending on the concentration of the target biomolecules, have
a different magnetization MC [25]:

f −3
d 
MC = M S  c  (1.4)
 ds 

with the fractal dimension of the micro-cluster f and the diameter of a single MNP
ds. Accordingly, T2 of the sample is commensurate with MC. In this respect, T2 is
linked with the amount of target upon nanoparticle agglomeration and attainable for
quantification. Unlike other sensing schemes, screening by NMR is rapid and low
cost as it is quasi label-free for the samples and immobilization-free for the trans-
ducers/electrodes. Such benefits render NMR-based detection as a promising solu-
tion for PoC applications. Although NMR is known for its relatively low sensitivity,
the MNP here provides inherent signal amplification to NMR since a single MNP
micro-cluster can affect billions of adjacent water molecules [26].
Conventionally NMR equipment are bulky and have limited applicability for
PoC diagnosis. Recently, researchers have been focusing on miniaturizing the mag-
net for NMR and migrating the modular and complex electronics into CMOS chips
[27–29]. With advanced circuit techniques to lessen the penalty of signal attenua-
tion induced by the compact magnet (<7.3 kg) as stated in (1.3), these micro-NMR
systems offer a trailblazing sensing method befitting more the PoC diagnosis.

1.4 Organization

The book is organized as below:

Chapter 2 reviews the state-of-the-art CMOS biosensors for in vitro diagnosis
[30]. The first session focuses on introducing different transducing mechanisms for
CMOS biosensors. According to the transducing mechanism, the CMOS biosensors
can be categorized into electrical based, optical based, magnetic based, mechanical
based, and NMR based. In the second session, in vitro diagnosis on different bio-
References 7

logical targets with CMOS biosensors and their applications is discussed.

Subsequently, the strengths and weaknesses of different sensing mechanisms for
these biosensors are compared in detail.
Chapter 3 illustrates the design and implementation of the palm-size micro-NMR
relaxometer with the electronic-automated digital microfluidic device for sample
management. Confounded by the limited inner space of the portable magnet, man-
agement of the sample under assay remains a critical issue for micro-NMR. Herein
the integration of micro-NMR with the digital microfluidic device is evinced to
facilitate the sample management and attain a comparable assay result to the con-
ventional approach [31–35].
Chapter 4 discloses the design of the micro-NMR relaxometer with B0-field sta-
bilization module [36, 37]. Ascribed to the temperature instability of the magnet,
calibration on either excitation frequency or Larmor frequency of the protons is nec-
essary to safeguard the operation of the micro-NMR relaxometer. Herein, a vertical
Hall sensor and relevant readout circuit are integrated with the micro-NMR trans-
ceiver to sense the B0-field variation of the magnet for calibrating the B0 field of the
portable magnet by injecting a current to its auxiliary coil. The closed-loop B0-field
stabilization here can suppress the variation of the B0 field and ease the operation.
Finally, Chap. 5 concludes the book and discusses the potential future works.

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Another random document with
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 79.

‘Why now my frends, for England fighte,’ I cryde:

‘Yf euer English hearts your noble breasts posseste,
I promise you to make them flinche, yf I may byde:
Mates follow me.’ Amongst my foes I rusht before the
rest:
‘O here come on,’ quoth I, ‘now fighte wee for the beste.’
And therewithall I vsde such courage, force, and
myghte:
As made my foes to fall, and soldiers fitly fighte.

80.

‘Yf we doe leese,’ quoth I, ‘the Frenche men shall not

gayne:
So if wee wyn, ’tis worth the while to keepe arraye:
Yf yee stand stiflye toet, wele make them peaze the
paine,
And leade with losse of lyuely lymmes the laude awaye.
Although they fearcely fighte, in hope vs all to slaye:
Lo, sixe to one they fall, and deade they lye:
Wee English men, in triomphe fight and honour dye.’

81.

With bloody broiles of war, the haplesse towne did

smoke,
The children sawe theire fathers deare, to bleede their
last:
The wyues bewayled muche the fatall stroke,
Which forste their husbands bleede, fall, dye so fast:
“Helas,” the weemen cryde, the woefull streets that past:
(When soe they sawe the channels bloody streame)
“What plague is this, that pesters so our reame?

82.
“Is no remorce of lyfe, but kill, kill, kill? helasse:
Kill, kill, the English cry, and valiantly they fighte:
What hap had wee to see these mischiues com to
passe?”
“Helas, le sang de nous amis, la mort, helas:”
The maydens cry: the widowes wayle, and aged
mourne,
With wringing hands vplift, and wish them selues
vnborne.

83.

Of vs one thousand Englishmen within the towne,

Sustaynde the force, the powre, and puissaunce of their
king:
And of the French that faught, wee beate three thousand
downe,
Wee slew no lesse, for all the nomber hee did bring:
Yf this vntrue shall seeme, discredite myne to ring,
A French historian writeing for them selues shall say:
Three thousand Frenchmen there, were slayne that
day.

84.

Four hundreth Englishmen that tyme were slayne in

fighte,
My selfe was one, with losse they wan the towne, perdie:
But if I might haue liude t’aue tride our righte,
With one for euery seuen, by ods as wee did dye:
I doubte not (so the rest, would done their partes as I:)
But that king Charles, his lords, nor all his men,
Should scarce haue tane the towne of Pontoise then.

85.

What neade I more debate of these thinges here:

In England was the faulte, though we did feele the
smarte:
While they at home, at bate and strife for honours were,
They lost abroade of Normandy the greater parte:
To thinke on this torments agayne my wounded harte,
That lords at home, should striue about the name,
And loose abroade their countrie’s weale and fame.

86.

Let English peeres abandon such contentious strife,

It hurtes the publique weale, decayes the state:
It reaues the yeares too soone of longer lyfe:
It freates the breste with ruste of baend debate:
It giues the checke to him that giues the mate:
Then thus I ende, that wight of all is bleste
Which liues in loue with God, his prince, and country
best.

87.

So Higins, yf thou write, how this my fall befell:

Place it in Baldwine’s Miroir with the reste:
From crazed scull sith here my mynde I tell,
Sith bleedeing hart these ruefull rymes expreste,
This mangled tale beseemes my person beste:
“Do so?” quoth hee “and let it passe euen thus:“
“Viuit” quoth I, “post funera virtus.”

Iohn Higins.[1881]
 [“This knight, my maisters,” quoth one, “came somwhat to late in
order.” “That is maruaile,” quoth maister Ferrers, “it seemes that hee
was forwarde enoughe in seruice.” “Yea,” quoth another, “hee came
the later home for that, and therefore wee must accept his cause.”
“How ere hee came,” quoth M. H.[1882] “hee sayes well, and like a
noble gentleman, as no doubt hee was.” “Hee should haue beene
placed,” quoth one, “after king Iames the first, king of Scots, of
whome wee spake in the yeare 1437.” “Now,” quoth I, “that you talke
of king Iames, I haue king Iames the fourth here, which was slayne
at the batayle of Brampton, or Floddon fielde, but hee is very
rude”[1883] “I like him,” quoth one, “the better: for if hee should bee
otherwise, it would not well beseeme his person, nor the place
whence he comes.” “Reade it,” quoth they, “as it is.” “Thinke then,”
quoth I, “that you see him standing all wounded, with a shafte in his
body, and, emongst other woundes, one geuen by a byll, both
deadly, to say in his rude and faithlesse maner as followeth.”]
[The lamentation of King James the
fourth, King of Scots, slayne at
Brampton, in the fiuthe yeare of King
Henry the eight, Anno Christi, 1513.
1.

As I lay musing, my selfe alone,

In minde not stable, but wauering here and there,
Morpheus my frend espyed mee anone,
And as hee was wont, whistered in mine eare:
Shortly conuyede I was, I wist not where:
Mine eyes were closed fast, I could not see:
I hearde a man crying[1884] sore, trembling for feare:
“Miserere mei Deus et salua mee.

2.

Miserere mei Deus:” oft hee did reporte,

With sorowfull sighes as euer man herde:
For sorowe and pity, I gan[1885] nere to resorte:
His sore exclamations made mee afferde:[1886]
Mine eyes opened, I sawe his grim bearde:[1887]
I knewe not verely, who it should bee:
Hee cryde, as hee had beene stickt with a swerde:
“Miserere mei Deus & salua mee.

3.

Of Scotland (hee sayde) late I was king,

With crowne on my[1888] head, and scepter in hand:
In wealth and honour, I wanted[1889] nothing:
In peaceable maner I ruled my land:
Full frendly and faythfull my subiects I fand:[1890]
Now am I exiled from life,[1891] land, and liberty:
King without realme, loe, now where I stand:
Miserere mei Deus & salua mee.

4.

Thus for my folly, I feele I doe smart,

Both law and nature doth me accuse
Of great vnkindnes: that I should take part
Against my brother, and his liege refuse:
I purposed war, yet I fayned truce:
Thus did I, Frenche king, for the loue of thee,
Inordinate affection so did mee abuse:
Miserere mei Deus & salua mee.

5.

All this, king Lewis, I suffred for thy sake,

Wo be to the time that euer I thee knewe:
For thee am I put in a[1892] sorowfull brake,
Thy wilfull appetite, doth me sore rewe:
This worlde is not stable, it chaungeth a newe:[1893]
Now am I bond, some time I was free:
Exiled from liberty, I am kept in a mewe:
Miserere mei Deus & salua mee.

6.

Moreouer for thee, and thy realme of Fraunce,

(Contrary to mine[1894] othe solemnly made)
Unto king Henry I made defiaunce,
To follow thine appetite was all the grace I hade:[1895]
In most cruell wise, I did his realme inuade:
I troubled his subiects, by land and by sea:
 My rewarde is no more, but the showle and spade:
[1896]
Miserere mei Deus & salua mee.

7.

For my wilfull periury, thus am I brought

From high degree, to the lowest of all:
Whom should I blame? I found that I sought,
By mine owne foly,[1897] I had a great fall:
Wherefore I feare mee, that now I shall
Haue payne long lasting, for mine iniquity:[1898]
Lord full of mercy yet to thee I call,
Miserere mei Deus & salua mee.

8.

Vanquished in fielde I was to the rebuke

Of mee and all my realme: to our immortall shame:[1899]
There faught agaynst mee neyther king, nor duke,
Prince, ne marquise, ne many lords of name:
One valiaunt earle, our power ouercame:
Yet were wee in nomber, to his one, three:
Lord whom thou fauourest, winneth[1900] the game:
Miserere mei Deus & salua mee.

9.

I was th’only[1901] author, of myne owne woe;

But yet I began it by[1902] wicked counsell,
Of my lords spirituall and temporall also:
Which for their merits in fielde with mee fell:
I was curst (in deede) the truth for to tell,
And could not (by falshoode) eyther thriue or thie:
To assist my brother’s foe I did not well,[1903]
Miserere mei Deus et salua mee.
 10.

Christe’s commaundements,[1904] I did all refuse:

The breatch of myne oathe,[1905] I did not regarde:
Therfore I am domed[1906] as faythlesse as the Jewes:
Sore is the sentence, and cruell is the swerde:[1907]
Excepte thy mercy helpe, O Lord, I am marde:
Saue mee; for whom thou suffredst on a tree,
To thy mercye I appeale for my sauegarde:
Miserere mei Deus & salua mee.

11.

Herafter (by mee) my successours may beware,

An ensample[1908] take by my wretched ruyne:
Lest in lykewyse they bee taken with[1909] the snare,
As I am nowe: and pay the[1910] lyke fyne:
Vanquished wee were, by[1911] power devyne:
For by manne’s power it seemed not to bee:
Here now I ly, in an homely shrine,
Miserere mei Deus & salua mee.

12.

I am a spectacle also in lyke case,

To the Frenche king, yf hee list to take heede,
I feare that hee cannot for lacke of grace,
The king[1912] and hee, bee not yet agreede:
Therefore let him looke, for a lyke speede,
As wee had that were of his leage and vnity,
I trow hee doth neither God[1913] loue, nor dreede,
Miserere mei Deus & salua mee.

13.

Who euer knew christian king in such a case,[1914]

 As[1915] I wretched creature that cannot haue
In churche or in[1916] churchyard any maner place,
Emong christen people to lye in a graue:
The earth mee abhorreth,[1917] all men mee depraue,
My frends forsake mee, and haue[1918] no pity,
The worlde taketh from mee all that hee mee gaue:
Miserere mei Deus & salua mee.

14.

There is no more now, I must take my leaue,

In this wretched worlde I may no longer dwell:
But one thing there is doth mee sore greaue,
I not where to rest, in heauen or in hell,
None else thereof but only God can tell:
Adieu, this worlde is full of vanity,
I may no longer be with thee, farewell:
Miserere mei Deus & salua mee.

15.

Farewell my queene, sweete lady Margaret:

Farewell my prince, with whom I vsde to play:
I wot not where wee shall together meete:
Farewell my lords and commons eke for aye:
Adieu, ye shall no ransom for mee pay:
Yet I beseeche you of your charity,
To the high Lorde mercifull that yee pray:[1919]
Miserere mei Deus & salua mee.”]

[“King Iames,” quoth one, “wil bee misliked for his Miserere.”
“No,” quod another, “hee cryes Peccaui.” “It is to late,” quoth he,
“there is no man that will like or beleeue him.” “Than,” quod M. H. “he
is still one and the same man: for in life he was neither well liked,
beleeued, nor trusted.” “Why than,” quoth one, “if hee speake as hee
was, let him passe as hee is, and if not, let him bee mended.”
“Mended,” quoth hee, “nay, hee is paste mending, hee is to olde: for
it seemes by the copy, that it was pende aboue fifty yeares agone, or
euen shortly after the death of the sayd king: for I found therewith, in
an olde hand, the copyes of the sayd king Iames’ letters sent unto
king Henry at Turwin, and the king’s aunsweres and letters sent to
him againe, with this lamentation ensuing them: and lastly the sayd
batayle of Floddon fielde, in such verse described, with the order of
the same, and the names of the noble men, knights, and gentlemen,
which serued at the same fielde.” “That would I faine heare,” quoth
one, “it were pity that such particulers should bee lost.” “They would,”
quoth another, “pleasure not only such as write our historyes, but
also encourage our countreymen well, to the like loyall seruice of
their prince, and especially those who should finde therein of their
parents or auncestours to haue bene praysed for valure.” “I pray
you,” quoth hee, “let us haue them.” “There they are,” quoth I, “but I
haue altered the verse, which wee call Intercalaris, because the rest
else would not haue beene well liked; but of the history I haue not
chaunged one word.”]
[The Bataile of Brampton, or Floddon
fielde, faught in the yeare of our
Redeemer 1513, and in the fiuth yeare
of the raygne of that victorious prince,
King Henry the eyght.
1.

O Rex regum in thy realme celestiall,

Glorified with ioyes of Gabriell’s company,
King Iames is dead, haue mercy on vs all:[1920]
For thou haste him prostrate so sodaynly,
(Which was our noble prince his enemy)
That vs to withstand hee had no might:
So thy helpe O Lord, preserude king Henrye’s right.

2.

Into England this prince prowdly did come,

With fourscore thousand in goodly aray:
And the castle of Norham first hee had won,
Prospering victoriously from day to day:
But agaynst him is gone the earle of Surrey,
With him manfully for to fight,
By the helpe of God and in his prince’s right.

3.

This noble earle full wisely hath wrought,

And with thirty thousand forwarde is gone:
After wisedom and pollicy wondrously[1921] hee sought,
How by the Scottish ordinaunce bee might well come:
Thereto helped well Basterd Heron,
On the Scots hee did harme both day and night,
So thy helpe O Lord, preserude our prince’s right.

4.

Our herald of armes to king Iemy did say:

“My lord of Surrey greetes[1922] you well by mee,
Maruayling greatly of this your array,
And what you make here in this countrey:
Peace you haue broken, and olde amity:
Wherefore if yee abide hee will with you fight,
By the helpe of God and in his prince’s right.”

5.

“Abyde:” hee sayde, “els were it great dishonour hye,

That a king crowned an earle durst not abide:
Yf Surrey bee so bolde to[1923] gieue battayle to mee,
I shall him tarry on Floddon hill side:
Open warre then soone was there cryde,”[1924]
And our doughty men were[1925] redily dight,
By the helpe of God, and in theyr prince’s right.

6.

S. Cutberd’s banner with the byshop’s men bolde,

In the vaunt garde[1926] forward fast did hye,
That royall relike more precious than golde,
And sir William Bowmer nere stoode it by:
“Adiuua pater,” then fast did they cry,
“Pray wee that God will graunt vs his might,
That wee may haue the powre to saue our prince’s
right.”
 7.

The lord Clifford and the lord Latimer also,

With the lord Couiers[1927] of the north countrey,
And the lord Scrope of Vpsalle forwarde did goe,
With the lorde Howarde admirall of the see,
Of noble hearte and courage good was hee,
As any went that time agaynst the Scots to fight,
By the helpe of God, and in theyr prince’s right.[1928]

8.

Sir William Percy and lorde Ogle both same,

And sir William Gascoyne, theyr cosin nere was hee:
The shryue of Yorkeshyre sir Ihon Eueringame,
And the nobles of Chesshyre in theyr degree:
The lord Dacres, and Basterd Heyron with heart free,
Which did harme the Scots by day and by night,
By the helpe of God, and in theyr prince’s right.

9.

Sir Edmond Haward of lusty franke courage

Boldly aduaunced himselfe eke in that stounde,
To the Scots our enemies he did great hurte and
domage,
Which were right greedy him and his bloud to confound:
But theyr mischieuous intent on themselues did rebound,
And many a deadly stroke on them there did light,
So the helpe of God preserude our prince’s right.

10.

The baron of Killerton, and both Astones were there,

With sir Iohn Bouthe, and many knightes moe:
Sir Iohn Gower and sir Walter Griffin drewe nere,
With sir Thomas Butler and maister Warcoppe also,
Sir Christopher Warde, and sir William Midylton both two,
 And sir William Maliuer, all did manly fight,
By the helpe of God, and in theyr prince’s right.

11.

In the mydle warde was the earle of Surrey,

That noble man, stoute, bolde, and hardy,[1929]
The father of wit wee call him may,[1930]
The deputy of England most trusty was hee:
With him lorde Scrope of Bolton, and sir George Darcye,
And sir Richard Maliuer with bucks heades bright,
By the helpe of God, and in their prince’s right.

12.

Sir Phillip Tilney was there ready and prest,

In the same warde, with all his mighty powre,
And sir Iohn Willowghby as ready as[1931] the best,
With sir Nicholas Aplyard his[1932] helpe, ayde, and
succour:
O what ioy was it to see that[1933] same howre,
How valiauntly our noble men with the Scots did fight,
By the helpe of God, and in theyr prince’s right.

13.

Yong sir William Gascoyne was there indeede,

With sir Richard Aldburgh, and sir Christofer Danebe,
Sir William Scarkell, and M. Frost’s help at neede,
With sir Raphe Ellarkar and M. Thomas Lee,
M. Raphe Beeston, and M. Hopton men might see
Full well, perdy,[1934] they quite themselues in that
fight,
By the helpe of God, and in theyr prince’s right.

14.
Sir Edward Stanley in the rear warde was hee,
A noble knight both wise and hardy,
With many a noble man of the west countrey,
And the whole powre of the earle of Darby,
With a right[1935] retinue of the bishop Elye,
And of Lankeshyre men manly[1936] did fight,
By the helpe of God, and in theyr prince’s right.

15.

Soone then the gunnes began[1937] a new play,

And the vaunt garde together are gone:
But our gunnes disseuered them out of aray,
And our bolde bilmen of them slewe many one:[1938]
So that of them scarce retourned[1939] none:
Thus were they punished by helpe of God almight:
So thy helpe O Lord, preseru’d our prince his right.

16.

Then they sought embushments, but with[1940] small

chere,
And in fowle[1941] maner brake theyr aray:
Yet some of our men by policy fled were,[1942]
That sawe[1943] king Iemy on the hill where he lay:
“They flee,” he sayes, “follow fast I you pray:”
But by that fit of flying[1944] wee wan the fight:
So the helpe of God preserude our prince’s right.

17.

To the earle of Surrey king Iemy is gone,

With as comly company as euer man did see:[1945]
Full boldly theyr big men agaynst vs did come
Downe the hill, with great myrth and melody:
And our men marked them to the Trinity,[1946]
 Beseeching him there to shew[1947] his might,
In theyr whole defence, and in theyr prince his right.

18.

The red lyon with his owne father’s bloud inclynate,

Came towards the white lyon both meeke and mylde,
And there by the hand of God he was prostrate,
By the helpe of th’eagle with her swadled chylde:
The buckesheads also the Scots has beguilde,
And with theyr grey goose wings doulfully them dight,
By the helpe of God, and in our prince his right.

19.

The moone that day did shine full bright,

And the luce head that day was full bent:
The red cressent did blinde the Scots sight,
And the ship with her ancre many Scots spent:[1948]
But, alas, the good white griffin was felde on Floddon hil,
Yet escape hee did, not vanquisht in the fight:[1949]
So thy helpe, O Lord, preserude our prince his right.

20.

The treyfell was true, and that did well appeare,

And boldly the great griffin vp the hill is gone:
The antlet did lace them with arrowes so nere,
That buffits the Scots bare, they lacked none:
The cinquefoile also was stedfast as the stone,
And slewe of the Scots like a worthy wight:[1950]
So thy helpe, O Lord, preserude our prince his right.

21.

The yong white lyon was angry in that stounde,

And with his merry mariners the myrth him made,
His bells rang lay[1951] couched in the grounde,
Whereof the Scots were[1952] right sore affrayde:
And round about rydeing euermore he sayde
Go to my fellowes all shalbe all or night,[1953]
By the helpe of God, wee saue our prince his right.

22.

The Cornish choughe did picke them in the face,

And the crab them blinded that they might not see:
They flewe and fell,[1954] they had none other grace,
With theyr new conquerour: but where now is hee,
Caryed in a cart, to his rebuke and his posterity,
And his bullies so bonnye are put all to[1955] flight:
So thy helpe, O Lord, preserude our prince his right.

23.

Of Scots lay[1956] slayne full xii thousande,

And xi earles, the sooth for to say,
Xiii lords, and three byshops as I vnderstande,
With two abbots, which haue learnde a new play,
They should haue bene at home for peace to pray:
Wherefore they were thus wise punished by right:
So thy helpe, O Lord, preserude our prince his right.

24.

Theyr ordinaunce is lost, and theyr royalty,

Wee haue theyr riches, God haue the prayseing:[1957]
What ech man[1958] would take, hee had his[1959] liberty:
Wherefore laude and honour to[1960] such a king,
From dolefull daunger vs so defending:[1961]
Hee has graunted vnto vs now his might,
And by his only ayde preserude our prince’s right.
 25.

O rex regum, ruler[1962] of vs all,

As thou for vs sufferedst[1963] thy passion,
Gieue the Scots grace, by king Iemye’s fall,
For to eschue for euer like transgression,[1964]
Preserue the red rose, and be his protection:
Laude, honour, prayse be vnto God almight,
Who thus suppreste our foes, preserud our prince’s
right.[1965]

26.

O yee noble lordes and knights victorius,

I you beseech to haue mee excused,
Your noble acts no better that I discusse,
And that my simple saying be not refused:
Where in any thing I haue mee misused,
I mee submit to your charitable correction:
And in this maner shall be my conclusion.

qd. Frauncis Dingley.[1966]]

 [The[1967] open bruite of princes falles and such as bare sway in
this realme, made mee poore haplesse woman (though once in great
place,) presume to shew my selfe emong that infortunate flock. And
making more haste then good speede, I appeared fyrst to one
Baldwine a minister and a preacher: whose function and calling
disdaynes to looke so lowe, as to searche the secrets of wanton
women, (though commonly a preacher with sufferaunce may rebuke
vice.) Wherefore I haue better bethought mee, and so doe sodaynly
appeale and appeare to some martiall man, who hath more
experience both in defending of women’s honour, and knowes
somwhat more of theyr conditions and qualityes: and the rather
because my tragedy was in question among some that would not
spare due commendation to the autor therof. I now appeare to him
that fyrst set mee forth, a writer of good continuaunce, and one that
dayly is exercised to set out both matter tragicall, and other
prophane histories and verses, whose name is Churchyard: hee
shall not only haue the fame of his owne worke (which no man can
deny)[1968] but shall likewise haue all the glory I can gieue him, if
hee lend mee the hearing of my woefull tale, a matter scarce fit for
woman’s shamefastnes to bewray. But since without blushing I haue
so long beene a talkatiue wench (whose words a world hath
delighted in) I will now goe on boldly with my audacious manner: and
so step I on the stage in my shrowdeing sheete as I was buried.]
How Shore’s wife, King Edward the
Fourth’s Concubine, was by King
Richard despoyled of all her goods,
and forced to doe open penaunce.
1.

Among the rest, by fortune ouerthrowne,

I am not least, that most may wayle her fate:
My fame and bruite abroade the world is blowne,
Who can forget, a thing thus done so late?
My great mischaunce, my fall, and heauy state,
Is such a marke, whereat each tongue doth shoote,
That my good name is pluckt vp by the roote.

2.

This wandring world bewitched mee with wyles,

And won my wits, with wanton sugred ioyes:
In fortune’s frekes, who trustes her when shee smiles,
Shall finde her false, and full of fickle toyes,
Her triumphs all, but fill our eares with noyse,
Her flattring giftes, are pleasures mixt with payne,
Yea, all her wordes are thunders threatning rayne.

3.

The fond desire that wee in glory set,

Doth thirle our hearts to hope in slipper hap,
A blast of pompe is all the fruite wee get,
And vnder that lies hid a sodayne clap: