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Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy

TL;DR: In this article, a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations.
Abstract: Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 104 wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

Summary (2 min read)

1. Introduction

  • The clinical validity of the assay was further demonstrated during the successful detection of BRAF and KRAS point mutations in colorectal, lung and melanoma cancer patients' tissue and plasma samples.
  • Results indicate excellent sensitivity of the method, comparable to that obtained with ddPCR, with a more simple and less expensive manner.

2. Materials and methods

  • The 150 MHz HFF-QCM acoustic array signals were monitored with a newly developed platform (AWS, S.L. Paterna, Spain).
  • The QCM sensors (AWS, S.L. Paterna, Spain), with a fundamental frequency of 5 MHz, were monitored using the Q-Sense E4 instrument (Biolin Scientific, Sweden) at the 7th overtone (35MHz).
  • Details on the device cleaning can be found in the Supplementary S1. 2.2 Acoustic detection of b-BSA and NAv. Protein samples were diluted in PBS pH=7.4 (Sigma-Aldrich).
  • Neutravidin (NAv-Invitrogen) (0.2 mg/mL) and biotinylated-BSA (b-BSA) (0.2 mg/mL) were applied on the device surface; 0.05 mg/mL of NAv was further applied on the b-BSA layer.
  • B-BSA was prepared as described in SI-S2.

2.5 BRAF V600E and KRAS G12D AS-PCR.

  • CfDNA was isolated from 2 mL of plasma for each sample via the QIAamp Circulating Nucleic Acid kit .
  • DdPCR was performed using the QX200 Droplet Digital PCR System (Bio-Rad), as previously described 25 and the KRAS G12/G13 and the BRAF V600 Screening Multiplex Kits (Bio-Rad).

3. Results and discussion

  • The protocol the authors developed for the detection of cancer point mutations in crude samples includes a step of amplification through liposomes; for this reason, they further monitored the binding of 200 nm diameter POPC liposomes on dsDNA in buffer.
  • ΔD values measured with the 150 MHz QCM array biochip during liposome attachment gave a higher dissipation value when liposomes were bound to the longer DNA (Fig. 2D ), in agreement with previous studies 24 .
  • Overall, the successful detection of proteins, DNA and liposomes with the 150 MHz acoustic array demonstrate the suitability of the new system for subsequent application for the development of molecular biology assays in crude samples.

Analytical performance of the AS-PCR / acoustic assay for the detection of point mutations using genomic DNA

  • Results indicated poor discrimination between the two (Fig. S1 ).
  • The real-time binding of the liposomes to the AS-PCR modified surface was specific, giving nearly zero dissipation change in the absence of the mt target (Fig. 3A ).
  • Based on these results (Fig. S3 ), a modified protocol for the AS-PCR including 45 cycles (1h) was established and used for the KRAS detection.
  • Regarding frequency change, the response was not as sensitive as the dissipation (Fig. S4 ), similarly to the BRAF V600E results.

Effect of the operating frequency.

  • To evaluate the efficiency of the 150 MHz acoustic biochip array towards the analysis of cancer point mutations the authors used the established 35MHz QCM sensor to perform the same assays and compare results.
  • Differences between the two devices would include the penetration depth inside the sensing solution (43 nm for the 150 MHz and 90 nm for the 35 MHz) and the sensitivity of frequency to the adsorbed mass; the latter according to the Sauerbrey equation scales up with the square of the fundamental resonator frequency fo.
  • In addition, differences in the size of the two QCM-devices, geometry of the flow cell and applied flow rate can affect the amount of immobilized target.
  • Based on Fig. 3E and F, the authors conclude that the two devices give the same dissipation response and LOD towards the detection of all tested mt:wt AS-PCR samples.
  • Moreover, the 35 MHz device also failed to detect the BRAF and KRAS AS-PCR samples upon their direct loading on the b-BSA/NAv coated sensor (Fig. S6 & S7 ).

Clinical validation of the method during the analysis of BRAF and KRAS mutations in tissue samples.

  • The final goal of this work was to assess the capability of the method to detect ctDNA carrying point mutations in patients' samples.
  • Results are summarized in Table 1 for Sanger sequencing, ddPCR and AS-PCR/acoustic detection.
  • Clinical validation during KRAS and BRAF mutations detection in patients' plasma samples.
  • In contrast to results obtained with tissue samples, in the case of plasma a change in ΔD was detected for the wt specimens, although significantly lower to that obtained for positive samples.
  • Comparing to the ddPCR, their method is more cost-effective since the authors use a standard thermocycler (<2.6K) and the acoustic platform (<10K), both an order of magnitude less expensive than a ddPCR machine (110K).

4. Conclusions

  • The authors report the successful application of a novel HFF-QCM 24-sensors array biochip for the acoustic detection of BRAF-V600E and KRAS-G12D point-mutations from patients' tissue and plasma samples upon enzymatic amplification.
  • AS-PCR was chosen for amplification due to the method's high specificity and already wide applicability on a routine basis.
  • The excellent sensitivity of the acoustic method (0.01-0.05% MAF), shown here to be comparable to that obtained with ddPCR but for a fraction of the cost and in a much faster manner, together with its technology-readiness level hold promise for fast adaptation in a clinical oncology lab for both tissue and liquid biopsy.
  • It is anticipated that the proposed methodology can become a promising tool to identify patients carrying somatic mutations in BRAF or KRAS genes.

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Figures (5)

Content maybe subject to copyright    Report

Acoustic array biochip combined with allele-specific PCR
for multiple cancer mutation analysis in tissue and liquid
biopsy
Nikoletta Naoumi
1,2
, Kleita Michaelidou
3
, George Papadakis
2
, Agapi E. Simaiaki
1
, Román
Fernández
5,6
, Maria Calero
6
, Antonio Arnau
5,6
, Achilleas Tsortos
2
, Sofia Agelaki
3,4
, Electra
Gizeli
*,1,2
1
Department of Biology, University of Crete, Vassilika Vouton, Heraklion, 70013, Greece
2
Institute of Molecular Biology and Biotechnology-FORTH, 100 N. Plastira Str., Heraklion 70013, Greece
3
Laboratory of Translational Oncology, School of Medicine, University of Crete, Heraklion, Vassilika Vouton, 70013
Crete, Greece
4
Department of Medical Oncology, University General Hospital of Heraklion, Vassilika Vouton, 71500 Crete, Greece.
5
Advanced Wave Sensors S. L., Algepser 24, 46988 Paterna, Spain
6
Centro de Investigación e Innovación en Bioingeniería, Universitat Politècnica de València, 46022 Valencia, Spain
ABSTRACT: Regular screening of cancerous point mutations is of importance to cancer management and
treatment selection. Although excellent techniques like next generation sequencing and droplet digital PCR are
available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented
where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal
Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point
mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS
G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%,
i.e., detection of 1 mutant copy of genomic DNA in an excess of 10
4
wild type molecules, was demonstrated;
moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with
a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma,
colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR,
demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%.
The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple
sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for
the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid
biopsy.
Keywords: High Fundamental Frequency QCM; dissipation monitoring; liposomes acoustic amplification; BRAF
and KRAS; molecular diagnostics; clinical oncology
.CC-BY-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460590doi: bioRxiv preprint

2
1. Introduction
Tumor tissue biopsy (TB) remains the gold standard method for cancer diagnosis and is an
important source for routine molecular profiling of hotspot somatic mutations. However, conventional
tissue biopsy still has a number of limitations that stem from its invasive nature. In many cases, is not
feasible to perform TB and is inappropriate for capturing intra-tumor heterogeneity, longitudinal profiling
of cancer biomarkers and monitoring of disease progression
1,2,3
.
Liquid biopsy (LB) is a promising non-invasive alternative strategy to tissue biopsy that allows the
study and characterization of different biomarkers such as cell-free DNA fragments originating from tumor
cells. Circulating tumor DNA (ctDNA) may carry the same genetic alterations as those of a primary tumor
and thus, can serve as a valuable diagnostic and prognostic tool to select targeted therapies and monitor
therapeutic response in real time
4
. While LB is simpler, faster and more cost-effective than TB, ctDNA
detection poses an analytical challenge since it is present in very small quantities (z/aM), is highly
fragmented and exists in samples with a background of abundant wild type (wt) cell free DNA, exhibiting a
ratio of mt:wt below 0.1 %
5
.
Generally, mutation analysis of ctDNA can be performed by two different approaches: next
generation sequencing (NGS) and PCR-based methods
6,7
. Although NGS has contributed considerably to
molecular diagnostics for clinical oncology, its application is restricted by the high cost, complexity and
slow turnaround time (up to 10 days); in addition, only a small number of labs are equipped with a NGS
facility. Real time PCR (qPCR) is also applied routinely for cancer molecular analysis. The limitations of
qPCR, compared to NGS-based methods, include the low sensitivity of detecting mutations in the presence
of wild type DNA (Mutant allele frequency; MAF of 10-20%)
4
, and the need for targeted mutation
analysis
8,9
. The Allele-Specific PCR (AS-PCR) and the third-generation droplet digital-PCR (ddPCR) are
able to detect 0.1% and 0.001% MAFs, respectively
7
. ddPCR has limitations including long droplet
processing times and high cost while AS-PCR, although more cost-effective, exhibits low sensitivity during
the amplification of cancer mutant (mt) targets in the presence of an excess of background wild-type (wt)
cell-free DNA.
Recently, DNA biosensors, including electrochemical
1012
, optical
13,14
and acoustic
1518
, have been
reported as an alternative means for the detection of point mutations. Moreover, advances in biochips and
nanotechnology have led to the development of enzymatic amplification-free protocols for ctDNA
7,19
. Such
examples include an electrochemical biochip combined with surface nanostructuring and a clutch/clamp
assay
20
; a plasmonic biosensor employing nanoparticles and a PNA-functionalized gold surface
21
; and a
piezoelectric plate sensor combined with fluorescent reporter microspheres
22
. Overall, the above works
present elegant examples of the application of biosensors in PCR-free clinical diagnostics, often achieving
impressive sensitivities similar to that of ddPCR
20
. Potential drawbacks include the (a) use for expensive
peptide nucleic acid (PNA) or locked nucleic acid (LNA) probes for the specific capturing of the mt target;
(b) requirement for hybridization steps and temperature control; (c) laborious surface-activation and
washing steps as well as nanoparticles synthesis and functionalization; and (d) low sensitivity, in cases
when low volumes of unpurified sample is used.
Because during the past decade LB has received tremendous attention, there is an urgent need for
the development of diagnostic tools for immediate application in the clinic
23
. Such tools should, ideally, be
rapid and simple to use; compatible to the routine methods currently employed in diagnostic labs for fast
adaptation; exhibit improved sensitivity and specificity; allow for multi-analysis; be robust; and, finally
provide a more economical solution to current methods. Inspired by the above needs, we developed a
method for the detection of point-mutations utilizing a novel High Fundamental Frequency Quartz Crystal
Microbalance (HFF-QCM) array-device with dissipation monitoring allowing multi-sample analysis
through the use of 24 resonators. Acoustic detection is coupled with AS-PCR for the amplification of 1-10
3
mt DNA targets carrying the BRAF V600E or KRAS G12D point mutation in a background of 10
4
wt
molecules. To combine AS-PCR with acoustic detection, we designed the assay so that the double strand
DNA (dsDNA) products could be directly immobilized on the device surface, obviating the need for a
hybridization step. Furthermore, we employed liposomes as acoustic signal enhancers in order to improve
the detection capability of the assay and detect down to 1 mutant copy in the initial sample in the presence
of 10
4
wild type DNAs.
The clinical validity of the assay was further demonstrated during the successful detection of
BRAF and KRAS point mutations in colorectal, lung and melanoma cancer patients’ tissue and plasma
samples. Results indicate excellent sensitivity of the method, comparable to that obtained with ddPCR, with
a more simple and less expensive manner.
.CC-BY-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460590doi: bioRxiv preprint

3
2. Materials and methods
2.1 Acoustic array and QCM sensor. The 150 MHz HFF-QCM acoustic array signals were monitored with
a newly developed platform (AWS, S.L. Paterna, Spain). The QCM sensors (AWS, S.L. Paterna, Spain),
with a fundamental frequency of 5 MHz, were monitored using the Q-Sense E4 instrument (Biolin
Scientific, Sweden) at the 7th overtone (35MHz). Details on the device cleaning can be found in the
Supplementary S1.
2.2 Acoustic detection of b-BSA and NAv. Protein samples were diluted in PBS pH=7.4 (Sigma-Aldrich).
Neutravidin (NAv-Invitrogen) (0.2 mg/mL) and biotinylated-BSA (b-BSA) (0.2 mg/mL) were applied on
the device surface; 0.05 mg/mL of NAv was further applied on the b-BSA layer. b-BSA was prepared as
described in SI-S2. Due to the different size of the HFF QCM and QCM devices and fluidics geometry, the
working volumes were V
150MHz
=60 μL and
V
35MHz
=200 μL.
2.3 HFF QCM detection of dsDNA and liposomes. dsDNA fragments of 21 bp, 50 bp 75 bp and 157 bp
were prepared according to
24
and applied (60 μL of 83 or 500 nM) to a NAv pre-coated array. 100 μL x 0.2
mg/mL of 200 nm of POPC liposomes (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), prepared by
extrusion as described before
24
, were added on the DNA surface.
2.4 Acoustic analysis of AS-PCR. For the acoustic detection of amplicons, the gold sensor surface was
modified with b-BSA followed by the addition of NAv (see above). A sample of 2.5 μL or 8 μL of the
BRAF or KRAS AS-PCR reaction respectively, diluted in a total volume of 20 μL, was loaded on the 150
MHz HFF QCM array (flow rate: 14 μL/min). Similarly, 2.5 μL or 10 μL of the BRAF or KRAS AS-PCR,
diluted in a total volume of 125 μL, was applied to the 35 MHz QCM device at a flow rate of 25 μL/min. In
both cases, a suspension of 0.2 mg/mL of 200 nm POPC liposomes was added at a volume of 100 μL (150
MHz) or 500 μL (35 MHz).
2.5 BRAF V600E and KRAS G12D AS-PCR. For the BRAF V600E, KAPA2G Fast HotStart ReadyMix
(KAPABIOSYSREMS) was mixed with 5 pmol of the allele-specific biotinylated reverse primer and with
5 pmol of the cholesterol modified forward primer in a total volume of 10 μL. For the KRAS G12D, 10
pmol of the mutation-specific biotinylated forward primer, 10 pmol of the cholesterol-modified reverse
primer and 1μL 20X SYBR® Green Ι Nucleic Acid Stain (Lonza) were mixed with the KAPA2G Fast
HotStart ReadyMix in a total volume of 20 μL. More information is provided in the SI-S3 and Table S1.
2.6 Sample collection. 21 FFPE tumor and 20 plasma samples were obtained from patients with various
cancer types at the University Hospital of Heraklion. The research protocol was approved by the
Institutional Ethics Committee of the University Hospital and all patients provided written informed
consent to participate in the study.
2.7 Sanger Sequencing and ddPCR for KRAS and BRAF-mutation analysis. gDNA from FFPE tissues, was
amplified by PCR using specific primer pairs for KRAS exon 2 and BRAF exon 15 (Table S1). Sequencing
reactions were performed using the Big Dye terminator V3.1 cycle sequencing kit (Applied Biosystems)
according to the manufacturer’s protocol. The products were then assessed by capillary electrophoresis on
an ABI3130 System and results were analyzed using Sequencing Analysis software v5.4 (Applied
Biosystems).
cfDNA was isolated from 2 mL of plasma for each sample via the QIAamp Circulating Nucleic Acid
kit (Qiagen). ddPCR was performed using the QX200 Droplet Digital PCR System (Bio-Rad), as
previously described
25
and the KRAS G12/G13 and the BRAF V600 Screening Multiplex Kits (Bio-Rad).
More information on ddPCR is provided in the SI-S4.
3. Results and discussion
Concept of combined AS-PCR / acoustic detection. The main objective of this work was to design a
methodology for liquid and tissue biopsy that could exhibit the high sensitivity of ddPCR using a less
cumbersome and more cost-effective method. The protocol we developed involves the specific
amplification of the point mutation via AS-PCR, followed by acoustic detection using a novel array biochip
device. The basic principle of the methodology is presented in Fig. 1A and B.
For the specific amplification of the mutant allele with AS-PCR, a set of primers
26
was used of which
the forward (Fw) amplifies both the mutant (mt) and wild type (wt) sequences while the reverse (Rv) only
the mt target; this is due to the Rv design which has the allele-specific (AS) nucleotide for the mt target in
the last position of the 3′-end. To further enhance Rv primer’s specificity, an artificial mismatched
nucleotide was introduced at the third from the 3′-end position. For the sake of the downstream acoustic
analysis, the Fw primer is modified with a cholesterol in its 5’-end and the Rv primer by a biotin (Fig. 1A).
.CC-BY-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460590doi: bioRxiv preprint

4
Following amplification, DNA fragments of 89 bp employing both a biotin and a cholesterol molecule in
the case of the mt target or only a cholesterol molecule in the case of the wt target are produced. The AS-
PCR reaction is then loaded directly on the NAv-modified acoustic biochip without prior purification. The
presence of NAv allows the immobilization of only the mt DNA-amplicons which carry the biotin. In order
to achieve the clinically relevant detection limits of few copies of mt target in the presence of larger
amounts of the wt (from 0.001-5%), ultrasensitive detection is necessary, even after AS-PCR. This
becomes even more significant if detection occurs directly inside the crude AS-PCR cocktail where issues
of non-specific binding become of concern. For this reason, a solution of POPC liposomes is injected and
captured by the immobilized products via the cholesterol-end of the mt amplified products (Fig. 1B).
Liposomes act as signal enhancers causing big changes in the acoustic signal leading to the detection of
immobilized DNA. This strategy has been shown by our group as well as others to be suitable for the
acoustic detection of Recombinase Polymerase Amplification (RPA) products
27
and single-base
mismatches
2830
.
Figure 1. Schematic illustration of the concept of the AS-PCR (A) and acoustic detection (B) for the BRAF V600E point-
mutation. (C) The acoustic array consists of 24 HFF-QCM sensors arranged in 6 lines of 4 sensors; (D) The PDMS flow cell
alone and (E) integrated with the array/PCB board; (F) Schematic representation of the liquid flow along the 6 lines and over
the 4 sensors.
Acoustic array biochip for multiple sample analysis. The HFF resonator array used in this work includes
24 miniaturized crystals integrated monolithically to a single substrate, in a layout of six rows with four
resonators/row (Fig. 2C). The HFF QCM array chip is ideal for high-throughput analysis
31
and low-volume
biosensing applications. Because the array is very small and fragile for direct handling during experiments,
it was mounted on a PCB (Fig. 2E); the latter also provides mechanical, electrical and thermal interface
between the acoustic wave device and the recording instrument. A gasket and a cell have been developed
and integrated with the PCB + array assembly (Fig. 2D & E). The flow cell device seals the microsensors
individually, so that it is possible to flow liquid in the desired direction over the sensor top surface without
affecting the array electrical connections placed on the bottom surface or interfere with the different lines
of sensors. During experimental conditions the liquid moves sequentially on each one of the four sensors in
the same row (Fig. 2F), running from the input to output. Each crystal has 0.3114 mm
2
active surface area
and 1.5 μL volume above the sensor. With the current flow set-up, six samples can be analyzed in a semi-
parallel way with the possibility to perform four tests per sample. More information on the array can be
found in Fernandez R.
32
.
Performance evaluation of the biochip array. Prior to the development of the combined AS-
PCR/acoustic biochip detection assay, we investigated the performance of the device in the presence of
pure samples and during the detection of protein, DNA and liposomes-binding. Overall, the principle of
operation of the HFF QCM resonator is the same to that of a typical acoustic device; briefly, the presence
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460590doi: bioRxiv preprint

5
of an analyte on the sensor surface affects the propagation characteristics of the acoustic wave, i.e. its
velocity and energy, which in turn are expressed as changes in frequency (ΔF) and dissipation (ΔD). ΔF
changes correlate with the amount of the deposited mass on the sensor
33
; ΔD changes reflect, among other
things, the viscoelastic properties of the surface-attached layer and the acoustic ratio (ΔD/ΔF) correlates to
the hydrodynamic properties
3436
or conformation
3740
in the case of discretely-bound molecules. We firstly
analyzed the device response during the absorption of proteins as well as the detection of dsDNA and
compared them to the response of the standard 35MHz QCM-D. Specifically, the physisorption of NAv and
b-BSA protein directly on gold was first recorded as well as the subsequent binding of NAv on a pre-
adsorbed layer of b-BSA. After each addition step, buffer rinsing was performed and the ΔF and ΔD
changes were measured at equilibrium and at surface saturation. Figure 2A & 2B shows that the relative
signal responses of all three proteins upon absorption/binding to the surface is the same for both devices.
Moreover, the array biochip was tested and found to give the expected linear relationship between the DNA
length and the acoustic ratio (ΔD/ΔF) during the binding of b-DNA molecules to a NAv-covered surface
through a single point (Fig. 2C)
37,39
.
The protocol we developed for the detection of cancer point mutations in crude samples includes a
step of amplification through liposomes; for this reason, we further monitored the binding of 200 nm
diameter POPC liposomes on dsDNA in buffer. We tested two different lengths of DNA i.e., 21 bp and 50
bp carrying a biotin at their 5’-end for immobilization to the surface and a cholesterol at their 3’-end for the
binding of liposomes. ΔD values measured with the 150 MHz QCM array biochip during liposome
attachment gave a higher dissipation value when liposomes were bound to the longer DNA (Fig. 2D), in
agreement with previous studies
24
. Overall, the successful detection of proteins, DNA and liposomes with
the 150 MHz acoustic array demonstrate the suitability of the new system for subsequent application for the
development of molecular biology assays in crude samples.
Figure 2. Comparison of ΔD (A) and ΔF values (Β) of NAv and b-BSA adsorption as well as NAv binding to pre-adsorbed
b-BSA on the 150 MHz and 35 MHz sensors; (C) Acoustic ratio (ΔD/ΔF) as a function of the length of b-DNA attached on a
NAv-modified surface; (C) Table of ΔD values at saturation during the binding of DNA (83 & 500 nM) followed by the
addition of liposomes (200 nm).
Analytical performance of the AS-PCR / acoustic assay for the detection of point mutations using
genomic DNA
(A) BRAF V600E. To determine the limit of detection (LOD) and sensitivity of the assay, mt genomic DNA
carrying the BRAF V600E point-mutation was mixed with wt DNA in a range from 0.01% to 10% (i.e.,
from 1:10
4
to 1:10 mt:wt). The mt:wt dilutions as well as the 100% (10
4
copies) wt genomic DNA (control)
were subjected to 55 cycles of AS-PCR (1h 40 min) followed by acoustic detection on the biochip array.
= 0.99
0
5
10
15
20
25
0 30 60 90 120 150 180
ΔD/ΔF (10
-9
/Hz)
DNA length (bp)
DNA
(nM)
DNA (bp)
ΔD
DNA
(10
-6
)
ΔD
POPC
(10
-6
)
83
21
23.5±2.9
324±53.0
50
39.4±8.6
346±70.0
500
21
83.3±4.8
375±31.6
50
147±34.0
439±39.0
C D
A
0
100
200
300
400
500
600
0
100
200
300
400
500
600
NAv
NAv on b-BSA
b-BSA
ΔF
35
(Hz)
ΔF
150
x10
2
(Hz)
150 MHz
35 MHz
Β
0
0.5
1
1.5
2
2.5
3
3.5
0
15
30
45
60
75
90
NAv
NAv on b-BSA
b-BSA
ΔD
35
(10
-6
)
ΔD
150
(10
-6
)
150 MHz
35 MHz
.CC-BY-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted September 17, 2021. ; https://doi.org/10.1101/2021.09.16.460590doi: bioRxiv preprint

Citations
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11 Jan 2023-Small
TL;DR: In this article , the major advances in the field of portable and semi-portable micro, nano, and multiplexed platforms for circulating cancer biomarkers (CBs) detection for the early diagnosis of cancer are highlighted.
Abstract: Liquid biopsy for the analysis of circulating cancer biomarkers (CBs) is a major advancement toward the early detection of cancer. In comparison to tissue biopsy techniques, liquid biopsy is relatively painless, offering multiple sampling opportunities across easily accessible bodily fluids such as blood, urine, and saliva. Liquid biopsy is also relatively inexpensive and simple, avoiding the requirement for specialized laboratory equipment or trained medical staff. Major advances in the field of liquid biopsy are attributed largely to developments in nanotechnology and microfabrication that enables the creation of highly precise chip-based platforms. These devices can overcome detection limitations of an individual biomarker by detecting multiple markers simultaneously on the same chip, or by featuring integrated and combined target separation techniques. In this review, the major advances in the field of portable and semi-portable micro, nano, and multiplexed platforms for CB detection for the early diagnosis of cancer are highlighted. A comparative discussion is also provided, noting merits and drawbacks of the platforms, especially in terms of portability. Finally, key challenges toward device portability and possible solutions, as well as discussing the future direction of the field are highlighted.

3 citations

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Journal ArticleDOI
TL;DR: It is shown that incubation temperature influences motility and limb bone growth in West African Dwarf crocodiles, producing altered limb proportions which may, influence post-hatching performance and provide new insights into how environmental factors can be integrated to influence cellular activity in growing bones and ultimately gross limb morphology, to generate phenotypic variation during prenatal development.
Abstract: Animals have evolved limb proportions adapted to different environments, but it is not yet clear to what extent these proportions are directly influenced by the environment during prenatal development. The developing skeleton experiences mechanical loading resulting from embryo movement. We tested the hypothesis that environmentally-induced changes in prenatal movement influence embryonic limb growth to alter proportions. We show that incubation temperature influences motility and limb bone growth in West African Dwarf crocodiles, producing altered limb proportions which may, influence post-hatching performance. Pharmacological immobilisation of embryonic chickens revealed that altered motility, independent of temperature, may underpin this growth regulation. Use of the chick also allowed us to merge histological, immunochemical and cell proliferation labelling studies to evaluate changes in growth plate organisation, and unbiased array profiling to identify specific cellular and transcriptional targets of embryo movement. This disclosed that movement alters limb proportions and regulates chondrocyte proliferation in only specific growth plates. This selective targeting is related to intrinsic mTOR (mechanistic target of rapamycin) pathway activity in individual growth plates. Our findings provide new insights into how environmental factors can be integrated to influence cellular activity in growing bones and ultimately gross limb morphology, to generate phenotypic variation during prenatal development.

1,786 citations

Journal ArticleDOI
TL;DR: Examining the mutational spectra of Ras isoforms curated from large-scale tumor profiling found that each isoform exhibits surprisingly distinctive codon mutation and amino-acid substitution biases, which were unexpected given that these mutations occur in regions that share 100% amino- acid sequence identity among the 3 isoforms.
Abstract: All mammalian cells express three closely related Ras proteins: H-Ras, K-Ras and N-Ras that promote oncogenesis when mutationally activated at codons 12, 13 or 61. Despite a high degree of similarity between the isoforms, K-Ras mutations are far more frequently observed in cancer and each isoform displays preferential coupling to particular cancer types. We have examined the mutation spectra of Ras isoforms curated from large-scale tumour profiling and found that each isoform exhibits surprisingly distinctive codon mutation and amino acid substitution biases. These were unexpected given that these mutations occur in regions that share 100% amino acid sequence identity between the three isoforms. Importantly, many of the mutational biases were not due to differences in exposure to mutagens because the patterns were still evident when compared within specific cancer types. We discuss potential genetic and epigenetic mechanisms together with isoform-specific differences in protein structure and signalling that may promote these distinct mutation patterns and differential coupling to specific cancers.

1,598 citations

Journal ArticleDOI
TL;DR: This Review will explore how tumour-associated mutations detectable in the blood can be used in the clinic after diagnosis, including the assessment of prognosis, early detection of disease recurrence, and as surrogates for traditional biopsies with the purpose of predicting response to treatments and the development of acquired resistance.
Abstract: Cancer is associated with mutated genes, and analysis of tumour-linked genetic alterations is increasingly used for diagnostic, prognostic and treatment purposes. The genetic profile of solid tumours is currently obtained from surgical or biopsy specimens; however, the latter procedure cannot always be performed routinely owing to its invasive nature. Information acquired from a single biopsy provides a spatially and temporally limited snap-shot of a tumour and might fail to reflect its heterogeneity. Tumour cells release circulating free DNA (cfDNA) into the blood, but the majority of circulating DNA is often not of cancerous origin, and detection of cancer-associated alleles in the blood has long been impossible to achieve. Technological advances have overcome these restrictions, making it possible to identify both genetic and epigenetic aberrations. A liquid biopsy, or blood sample, can provide the genetic landscape of all cancerous lesions (primary and metastases) as well as offering the opportunity to systematically track genomic evolution. This Review will explore how tumour-associated mutations detectable in the blood can be used in the clinic after diagnosis, including the assessment of prognosis, early detection of disease recurrence, and as surrogates for traditional biopsies with the purpose of predicting response to treatments and the development of acquired resistance.

1,424 citations

Journal ArticleDOI
TL;DR: How different forms of liquid biopsies can be exploited to guide patient care and should ultimately be integrated into clinical practice is examined, focusing on liquid biopsy of ctDNA — arguably the most clinically advanced approach.
Abstract: During cancer progression and treatment, multiple subclonal populations of tumour cells compete with one another, with selective pressures leading to the emergence of predominant subclones that replicate and spread most proficiently, and are least susceptible to treatment. At present, the molecular landscapes of solid tumours are established using surgical or biopsy tissue samples. Tissue-based tumour profiles are, however, subject to sampling bias, provide only a snapshot of tumour heterogeneity, and cannot be obtained repeatedly. Genomic profiles of circulating cell-free tumour DNA (ctDNA) have been shown to closely match those of the corresponding tumours, with important implications for both molecular pathology and clinical oncology. Analyses of circulating nucleic acids, commonly referred to as 'liquid biopsies', can be used to monitor response to treatment, assess the emergence of drug resistance, and quantify minimal residual disease. In addition to blood, several other body fluids, such as urine, saliva, pleural effusions, and cerebrospinal fluid, can contain tumour-derived genetic information. The molecular profiles gathered from ctDNA can be further complemented with those obtained through analysis of circulating tumour cells (CTCs), as well as RNA, proteins, and lipids contained within vesicles, such as exosomes. In this Review, we examine how different forms of liquid biopsies can be exploited to guide patient care and should ultimately be integrated into clinical practice, focusing on liquid biopsy of ctDNA - arguably the most clinically advanced approach.

1,292 citations

Journal ArticleDOI
TL;DR: The potential of liquid biopsies is highlighted by studies that show they can track the evolutionary dynamics and heterogeneity of tumours and can detect very early emergence of therapy resistance, residual disease and recurrence, but their analytical validity and clinical utility must be rigorously demonstrated before this potential can be realized.
Abstract: Precision oncology seeks to leverage molecular information about cancer to improve patient outcomes. Tissue biopsy samples are widely used to characterize tumours but are limited by constraints on sampling frequency and their incomplete representation of the entire tumour bulk. Now, attention is turning to minimally invasive liquid biopsies, which enable analysis of tumour components (including circulating tumour cells and circulating tumour DNA) in bodily fluids such as blood. The potential of liquid biopsies is highlighted by studies that show they can track the evolutionary dynamics and heterogeneity of tumours and can detect very early emergence of therapy resistance, residual disease and recurrence. However, the analytical validity and clinical utility of liquid biopsies must be rigorously demonstrated before this potential can be realized.

809 citations

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Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Acoustic array biochip combined with allele-specific pcr for multiple cancer mutation analysis in tissue and liquid biopsy" ?

Here a new approach is presented where allele-specific PCR ( AS-PCR ) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance ( HFF-QCM ) array biosensor for the amplification and detection, respectively, of cancer point mutations.