Single‐Molecule Bioelectronic Label‐Free Assay of both Protein and Genomic Markers of Pancreatic Mucinous Cysts’ in Whole Blood Serum

Macchia, Eleonora; Sarcina, Lucia; Driescher, Caroline; Gounani, Zahra; Tewari, Amit; Österbacka, Ronald; Palazzo, Gerardo; Tricase, Angelo; Vajna, Z.M.K.; Viola, Fabrizio Antonio; Modena, Francesco; Caironi, Mario; Torricelli, Fabrizio; Espósito, Irene; Torsi, Luisa

doi:10.1002/aelm.202100304

Cited by 24 publications

(35 citation statements)

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“…3-Mercaptopropionic acid (3-MPA), 11-mercaptoundecanoic acid (11-MUA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and the N -hydroxysulfosuccinimide sodium salt (Sulfo-NHS) were obtained from Merck Millipore (now MilliporeSigma). KRAS G12D (product number C9745(D07)) as target oligonucleotide, a biotinylated-KRAS G12D fwd (b-KRAS) (product number C9745(E03)) as biorecognition element and KRAS G12D 1MM (product number C9745(D12)) (one mismatch in correspondence of 14th nucleic acid base, used as negative control) were provided by Invitrogen-Thermo Fisher Scientific (oligonucleotides sequences are specified in previous reports) [ 10 ]. Human serum samples were obtained from blood samples collected from different healthy donor (age range 18–60 years, Research Donors Ltd of London [UK]) and diluted 1:4 with PBS and successively centrifuged at 10,000× g for 5 min, before the 3D SiMoT assay.…”

Section: Methodsmentioning

confidence: 99%

“…The 3D SiMoT device with an inkjet-printed organic semiconductor and lateral gate on plastic foil was stabilized before performing sensing experiments, according to the protocol defined and validated elsewhere [ 10 , 24 ]. The sensing measurements were performed according to the following protocol.…”

Section: Methodsmentioning

confidence: 99%

“…low vs. high risk). The addition of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which has been rarely investigated so far [ 10 ]. For instance, MUC1 is a glycoprotein almost exclusively expressed by high-risk pancreatic cysts as well as in cells of pancreatic cancer itself [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

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A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

et al. 2022

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Early diagnosis in a premalignant (or pre-invasive) state represents the only chance for cure in neoplastic diseases such as pancreatic-biliary cancer, which are otherwise detected at later stages and can only be treated using palliative approaches, with no hope for a cure. Screening methods for the purpose of secondary prevention are not yet available for these cancers. Current diagnostic methods mostly rely on imaging techniques and conventional cytopathology, but they do not display adequate sensitivity to allow valid early diagnosis. Next-generation sequencing can be used to detect DNA markers down to the physical limit; however, this assay requires labeling and is time-consuming. The additional determination of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which holds the potential to improve diagnostic accuracy. Moreover, the possibility to detect biomarkers in blood serum offers the advantage of a noninvasive diagnosis. In this study, both the DNA and protein markers of pancreatic mucinous cysts were analyzed in human blood serum down to the single-molecule limit using the SiMoT (single-molecule assay with a large transistor) platform. The SiMoT device proposed herein, which exploits an inkjet-printed organic semiconductor on plastic foil, comprises an innovative 3D-printed sensing gate module, consisting of a truncated cone that protrudes from a plastic substrate and is compatible with standard ELISA wells. This 3D gate concept adds tremendous control over the biosensing system stability, along with minimal consumption of the capturing molecules and body fluid samples. The 3D sensing gate modules were extensively characterized from both a material and electrical perspective, successfully proving their suitability as detection interfaces for biosensing applications. KRAS and MUC1 target molecules were successfully analyzed in diluted human blood serum with the 3D sensing gate functionalized with b-KRAS and anti-MUC1, achieving a limit of detection of 10 zM and 40 zM, respectively. These limits of detection correspond to (1 ± 1) KRAS and (2 ± 1) MUC1 molecules in the 100 μL serum sample volume. This study provides a promising application of the 3D SiMoT platform, potentially facilitating the timely, noninvasive, and reliable identification of pancreatic cancer precursor cysts. Graphical abstract

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

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“…Indeed, a more relevant role has been hypothesised when implemented in electrolyte gated organic field effect transistors (EGOFETs); biosensors, described by Macchia et al, exhibited an LOD at the single-molecule level, with 60 zM in 100 µL of sample volume [ 48 , 49 , 50 ], considering a millimetre-sized sensing interface modified with 10 11 –10 12 bioreceptor cm −2 ( wide-field approach) [ 48 , 49 ]. Herein, diffuse interchain hydrogen bondings between amides and carboxylic groups within the SAM monolayer play a key role in the sensing mechanism, hypothesising a signal amplification, which is fundamental for single-binding event detection, which is still under investigation.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical and X-ray Photoelectron Spectroscopy Surface Characterization of Interchain-Driven Self-Assembled Monolayer (SAM) Reorganization

et al. 2022

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Herein, we report a combined strategy encompassing electrochemical and x-ray photoelectron spectroscopy (XPS) experiments to investigate self-assembled monolayer (SAM) conformational reorganization onto an electrode surface due to the application of an electrical field. In particular, 3-mercaptopriopionic acid SAM (3MPA SAM) modified gold electrodes are activated with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHSS) (EDC-NHSS) mixture by shortening the activation time, from 2 h to 15/20 min, labelled as Protocol-A, -B and -C, respectively. This step, later followed by a deactivation process with ethanolamine (EA), plays a key role in the reaction yields (formation of N-(2-hydroxyethyl)-3-mercaptopropanamide, NMPA) but also in the conformational rearrangement observed during the application of the electrical field. This study aims at explaining the high performance (i.e., single-molecule detection at a large electrode interface) of bioelectronic devices, where the 3MPA-based SAM structure is pivotal in achieving extremely high sensing performance levels due to its interchain interaction. Cyclic voltammetry (CV) experiments performed in K4Fe(CN)6:K3Fe(CN)6 for 3MPA SAMs that are activated/deactivated show similar trends of anodic peak current (IA) over time, mainly related to the presence of interchain hydrogen bonds, driving the conformational rearrangements (tightening of SAMs structure) while applying an electrical field. In addition, XPS analysis allows correlation of the deactivation yield with electrochemical data (conformational rearrangements), identifying the best protocol in terms of high reaction yield, mainly related to the shorter reaction time, and not triggering any side reactions. Finally, Protocol-C’s SAM surface coverage, determined by CV in H2SO4 and differential pulse voltammetry (DPV) in NaOH, was 1.29 * 1013 molecules cm−2, being similar to the bioreceptor surface coverage in single-molecule detection at a large electrode interface.

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Stable and Solution‐Processable Cumulenic sp‐Carbon Wires: A New Paradigm for Organic Electronics

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has boosted the field of large-area flexible and printed electronics. These advances have enabled a plethora of applications such as organic light-emitting diodes, [1,2] organic photovoltaics, [3,4] organic thermoelectrics, [5,6] organic field-effect transistors (OFETs), [7][8][9][10] organic (bio)sensors, [11][12][13] and neuromorphic devices. [14,15] In this context, organic field-effect transistors (OFETs) are not only relevant for their direct technological application, but they also represent an ideal test-bed to investigate thin-film electrical properties. Organic semiconductors are typically classified in two main families, namely conjugated polymers and small molecules. The former, polymers, are particularly appealing as a result of their solution processability, and OFETs with charge mobility above the standard for hydrogenated amorphous silicon (0.5-1 cm 2 V −1 s −1 ) have been extensively reported. [16] The latter, small molecules, are prone to arrange in ordered molecular crystals, and through several years of chemical tailoring and fine tuning of the films processing, small-molecule OFETs with field-effect mobility >10 cm 2 V −1 s −1 have been achieved. [17][18][19] The chemical root of the π-conjugation of these materials is associated with the sp 2 -hybridization of carbon atoms in their backbone. This peculiar trait is also common to Solution-processed, large-area, and flexible electronics largely relies on the excellent electronic properties of sp 2 -hybridized carbon molecules, either in the form of π-conjugated small molecules and polymers or graphene and carbon nanotubes. Carbon with sp-hybridization, the foundation of the elusive allotrope carbyne, offers vast opportunities for functionalized molecules in the form of linear carbon atomic wires (CAWs), with intriguing and even superior predicted electronic properties. While CAWs represent a vibrant field of research, to date, they have only been applied sparingly to molecular devices. The recent observation of the field-effect in microcrystalline cumulenes suggests their potential applications in solution-processed thin-film transistors but concerns surrounding the stability and electronic performance have precluded developments in this direction. In the present study, ideal field-effect characteristics are demonstrated for solution-processed thin films of tetraphenyl[3]cumulene, the shortest semiconducting CAW. Films are deposited through a scalable, large-area, meniscus-coating technique, providing transistors with hole mobilities in excess of 0.1 cm 2 V −1 s −1 , as well as promising operational stability under dark conditions. These results offer a solid foundation for the exploitation of a vast class of molecular semiconductors for organic electronics based on sp-hybridized carbon systems and create a previously unexplored paradigm.

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Single‐Molecule Bioelectronic Label‐Free Assay of both Protein and Genomic Markers of Pancreatic Mucinous Cysts’ in Whole Blood Serum

Cited by 24 publications

References 50 publications

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

Electrochemical and X-ray Photoelectron Spectroscopy Surface Characterization of Interchain-Driven Self-Assembled Monolayer (SAM) Reorganization

Stable and Solution‐Processable Cumulenic sp‐Carbon Wires: A New Paradigm for Organic Electronics

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