Zeptomole Level Research Articles

Effect of NiAl alloy microparticles deposited in flexible SERS substrates on the limit of detection of rhodamine B molecules.

Flexible-SERS (FSERS) substrates were fabricated by depositing Ni64Al36(NiAl)-alloy-microparticles and/or spherical Ag-NPs (sizes of 10-40 nm) on recycled plastics, which had an aluminum layer on their surface. First, FSERS substrates made of Al + Ag-NPs and an area of 1 cm2 were used to detect rhodamine B (RhB) molecules. The limit-of-detection (LOD) for RhB was 8.35 × 10-22 moles (∼503 molecules), and the enhancement factor (EF) was 3.11 × 1015. After adding NiAl-microparticles to the substrate, the LOD decreased to 8.35 × 10-24 moles (∼5 molecules) and the EF was increased to 2.05 × 1017. Such EF values were calculated with respect to substrates made only with Al + NiAl-alloy (without Ag-NPs), which did not show any Raman signal. Other FSERS substrates were made with graphene-layer + Ag-NPs or graphene-layer + NiAl-alloy + Ag-Nps, and the best LOD and EF values were 8.35 × 10-22 moles and 6.89 × 1015, respectively. Overall, combining the Ag-NPs and NiAl-alloy microparticles allowed for the zeptomole detection of RhB. This was possible due to the formation of Ag aggregates around the alloy microparticles, which enhanced the number of hotspots. If no alloy is present in the FSERS substrates, the detection of RhB is lowered. Overall, we presented a low-cost FSERS substrate that does not require expensive Au films or Au-NPs (as previously reported) to detect RhB at the zeptomole level.

Expanding chiral metamaterials for retrieving fingerprints via vibrational circular dichroism

Circular dichroism (CD) spectroscopy has been widely demonstrated for detecting chiral molecules. However, the determination of chiral mixtures with various concentrations and enantiomeric ratios can be a challenging task. To solve this problem, we report an enhanced vibrational circular dichroism (VCD) sensing platform based on plasmonic chiral metamaterials, which presents a 6-magnitude signal enhancement with a selectivity of chiral molecules. Guided by coupled-mode theory, we leverage both in-plane and out-of-plane symmetry-breaking structures for chiral metamaterial design enabled by a two-step lithography process, which increases the near-field coupling strengths and varies the ratio between absorption and radiation loss, resulting in improved chiral light-matter interaction and enhanced molecular VCD signals. Besides, we demonstrate the thin-film sensing process of BSA and β-lactoglobulin proteins, which contain secondary structures α-helix and β-sheet and achieve a limit of detection down to zeptomole level. Furthermore, we also, for the first time, explore the potential of enhanced VCD spectroscopy by demonstrating a selective sensing process of chiral mixtures, where the mixing ratio can be successfully differentiated with our proposed chiral metamaterials. Our findings improve the sensing signal of molecules and expand the extractable information, paving the way toward label-free, compact, small-volume chiral molecule detection for stereochemical and clinical diagnosis applications.

Advances in single-molecule junctions as tools for chemical and biochemical analysis.

The development of miniaturized electronics has led to the design and construction of powerful experimental platforms capable of measuring electronic properties to the level of single molecules, along with new theoretical concepts to aid in the interpretation of the data. A new area of activity is now emerging concerned with repurposing the tools of molecular electronics for applications in chemical and biological analysis. Single-molecule junction techniques, such as the scanning tunnelling microscope break junction and related single-molecule circuit approaches have a remarkable capacity to transduce chemical information from individual molecules, sampled in real time, to electrical signals. In this Review, we discuss single-molecule junction approaches as emerging analytical tools for the chemical and biological sciences. We demonstrate how these analytical techniques are being extended to systems capable of probing chemical reaction mechanisms. We also examine how molecular junctions enable the detection of RNA, DNA, and traces of proteins in solution with limits of detection at the zeptomole level.

Ultrathin Suspended Chiral Metasurfaces for Enantiodiscrimination (Adv. Mater. 37/2022)

Metasurfaces A self-aligned suspended chiral bilayer metasurface via single-step electron-beam lithography is reported by Tun Cao, Yan Jun Liu, and co-workers in article number 2203956. A huge optical chirality of 221° μm−1 is achieved by the free-standing metasurfaces with overall thickness of 100 nm. The bilayer metasurface demonstrates label-free discrimination of chiral molecules at zeptomole level, with sensitivity orders of magnitude larger than conventional circular dichroism spectroscopy.

Ultrathin Suspended Chiral Metasurfaces for Enantiodiscrimination.

Chiral metasurfaces can exhibit a strong circular dichroism, but it is limited by the complicated fabrication procedure and alignment errors. Here, a new type of self-aligned suspended chiral bilayer metasurface with only one-step electron beam lithography exposure is demonstrated. A significant optical chirality of 221°µm-1 can be realized using suspended metasurfaces with a thickness of 100nm. Furthermore, this study experimentally demonstrates that such a structure is capable of label-free discrimination of the chiral molecules at zeptomole level, exhibiting a much higher sensitivity (orders of magnitude) compared to the conventional circular dichroism spectroscopy. The fundamental principles for chiral sensing using molecules-metasurfaces interactions are explored. Benefiting from the giant chiroptical response, the proposed metadevice may offer promising applications for ultrathin circular polarizers, chiral molecular detectors, and asymmetry information processing.

Zeptomole Detection of an Enzyme by a Simple Colorimetric Method.

An enzyme immunoassay, in which an enzyme (e.g., alkaline phosphatase, ALP) is conjugated with an antibody, is a precise and simple protein detection method. Precise measurements of enzymes at low concentrations allow for ultrasensitive protein detection. The application of a phosphorylated substrate to ALP, followed by using a dephosphorylated substrate in thionicotinamide-adenine dinucleotide cycling, provides a simple and precise quantification of ALP. We describe a protocol for detecting ALP at the zeptomole level using a simple colorimetric method.

Sub-zeptomole Detection of Biomarker Proteins Using a Microfluidic Immunoarray with Nanostructured Sensors.

We report here a low-cost electrochemical immunoarray with unprecedented sensitivity in the sub-zeptomole range with up to 5 log-decades dynamic range for accurate, multiplexed protein determinations. The microfluidic array features eight carbon sensors coated with a dense layer of 5 nm gold-nanoparticles derivatized with primary antibodies. Analyte proteins are captured by secondary antibody-poly-HPR (horseradish peroxidase) bioconjugates containing 400 HRP enzyme labels, with amplified amperometric peaks developed using H2O2 activator and hydroquinone mediator. Prostate cancer biomarkers prostate specific antigen (PSA), vascular endothelial growth factor-D (VEGF-D), ETS-related gene protein (ERG), and insulin-like growth factor-1 (IGF-1) were measured simultaneously with sub-fg/mL LODs (0.08-0.22 zmol). These proteins were determined in serum of postprostatectomy cancer patients which had much lower levels than prostate cancer patients without surgery. This immunoassay protocol makes thousands of low-abundance proteins accessible to quantitative measurements down to zeptomole levels.

In Liquid Infrared Scattering Scanning Near-Field Optical Microscopy for Chemical and Biological Nanoimaging.

Imaging biological systems with simultaneous intrinsic chemical specificity and nanometer spatial resolution in their typical native liquid environment has remained a long-standing challenge. Here, we demonstrate a general approach of chemical nanoimaging in liquid based on infrared scattering scanning near-field optical microscopy (IR s-SNOM). It is enabled by combining AFM operation in a fluid cell with evanescent IR illumination via total internal reflection, which provides spatially confined excitation for minimized IR water absorption, reduced far-field background, and enhanced directional signal emission and sensitivity. We demonstrate in-liquid IR s-SNOM vibrational nanoimaging and conformational identification of catalase nanocrystals and spatio-spectral analysis of biomimetic peptoid sheets with monolayer sensitivity and chemical specificity at the few zeptomole level. This work establishes the principles of in-liquid and in situ IR s-SNOM spectroscopic chemical nanoimaging and its general applicability to biomolecular, cellular, catalytic, electrochemical, or other interfaces and nanosystems in liquids or solutions.

Colocalized Particle Counting Platform for Zeptomole Level Multiplexed Quantification.

For multiplexed detection, it is important yet challenging to simultaneously meet the requirement of sensitivity, throughput, and implementation convenience for practical applications. Using the detection of DNAs and miRNAs for illustration, we present a colocalized particle counting platform that can realize the separation-free multiplexed detection of 6 nucleic acid targets with a zeptomole sensitivity and a dynamic range of up to 5 orders of magnitude. The presence of target induces the formation of a sandwich nanostructure via hybridization; thus, there is an occurrence of colocalization of two microbeads with two different colors. The sequence specific coding is realized by an arbitrary combination of two fluorescence channels with different emitting colors. The platform presents robustness in detecting multiple nucleic acid targets with a minimal cross talk and matrix effect as well as the ability to distinguish the specific miRNA from members of the same family. The results of simultaneous detection of 3 miRNAs in 3 different cell lines present straight consistency with that of the standard qRT-PCR. This platform can be adapted to other multiplexing designs such as the "turn-off" mode, in which the proportion of colocalized microbeads is decreased due to the strand-displacement reaction initiated by the specific target. This separation-free platform offers the possibility to achieve the on-site multiplexed detection with compatibility to different experimental designs and extensibility to other signal sources for enumeration.

Microfluidic exponential rolling circle amplification for sensitive microRNA detection directly from biological samples

There is an urgent need of sensitive bioanalytical platforms for sensitive and precise quantification of low-abundance microRNA targets in complex biological samples, including liquid biopsies of tumors. Many of current miRNA biosensing methods require laborious sample pretreatment procedures, including extraction of total RNA, which largely limits their biomedical and clinical applications. Herein we developed an integrated Microfluidic Exponential Rolling Circle Amplification (MERCA) platform for sensitive and specific detection of microRNAs directly in minimally processed samples. The MERCA system integrates and streamlines solid-phase miRNA isolation, miRNA-adapter ligation, and a dual-phase exponential rolling circle amplification (eRCA) assay in one analytical workflow. By marrying the advantages of microfluidics in leveraging bioassay performance with the high sensitivity of eRCA, our method affords a remarkably low limit of detection at <10 zeptomole levels, with the ability to discriminate single-nucleotide difference. Using the MERCA chip, we demonstrated quantitative detection of miRNAs in total RNA, raw cell lysate, and cell-derived exosomes. Comparing with the parallel TaqMan RT-qPCR measurements verified the adaptability of the MERCA system for detection of miRNA biomarkers in complex biological materials. In particular, high sensitivity of our method enables direct detection of low-level exosomal miRNAs in as few as 2 × 106 exosomes. Such analytical capability immediately addresses the unmet challenge in sample consumption, a key setback in clinical development of exosome-based liquid biopsies. Therefore, the MERCA would provide a useful platform to facilitate miRNA analysis in broad biological and clinical applications.

Zeptomole Biosensing of DNA with Flexible Selectivity Based on Acoustic Levitation of a Single Microsphere Binding Gold Nanoparticles by Hybridization.

A novel scheme for DNA sensing at the zeptomole level is presented, based on the levitation of a single microsphere in a combined acoustic-gravitational (CAG) field. The levitation of a microsphere in the field is predominantly determined by its density. Capture and reporter probe DNAs are anchored on poly(methyl methacrylate) microsphere (PMMA) and gold nanoparticles (AuNPs), respectively, and a target DNA induces the binding of AuNPs on PMMA. This interparticle sandwich DNA-hybridization induces density increase in PMMA, which is detected as a shift in the levitation coordinate in the CAG field. The reporter DNAs are designed based on base-pair (bp) number selectivity, which is evaluated using direct interparticle hybridization between DNA-bound PMMA and complementary DNA-anchored AuNPs. Interestingly, the bp-number selectivity can be enlarged by lowering the reactant concentrations. Thus, the threshold bp, at which no density change is detected, can be adjusted by controlling the reactant concentrations. This strategy is extended to the sensing of HIV-2 DNA and single nucleotide polymorphism (SNP) detection of the KRAS gene by sandwich hybridization. In SNP detection, the present method selectively distinguishes wild-type DNA from mutant DNA differing by one nucleotide in the 21-nucleotide sequence by optimizing the lengths of probe DNAs and particle concentrations. This approach allows the detection of 1000 DNA molecules.

Chirality detection of enantiomers using twisted optical metamaterials

Many naturally occurring biomolecules, such as amino acids, sugars and nucleotides, are inherently chiral. Enantiomers, a pair of chiral isomers with opposite handedness, often exhibit similar physical and chemical properties due to their identical functional groups and composition, yet show different toxicity to cells. Detecting enantiomers in small quantities has an essential role in drug development to eliminate their unwanted side effects. Here we exploit strong chiral interactions with plasmonic metamaterials with specifically designed optical response to sense chiral molecules down to zeptomole levels, several orders of magnitude smaller than what is typically detectable with conventional circular dichroism spectroscopy. In particular, the measured spectra reveal opposite signs in the spectral regime directly associated with different chiral responses, providing a way to univocally assess molecular chirality. Our work introduces an ultrathin, planarized nanophotonic interface to sense chiral molecules with inherently weak circular dichroism at visible and near-infrared frequencies.

Thiol-ene micropillar array electrospray ionization platform for zeptomole level bioanalysis.

A micropillar array electrospray ionization (μPESI) platform fabricated from thiol-enes with 56 individual polyethylene glycol coated μPESI chips for bioanalytical mass spectrometry is introduced. Bioanalysis capability is shown by measurement of a protein, a protein digest and a cell lysate sample. The thiol-ene polyethylene glycol (PEG) coated μPESI chip allows the use of a wide range of aqueous-organic solvent compositions and provides a detection limit at 60 zeptomole level (6 × 10-20 mol) for a peptide standard.

Signal-on electrochemical detection of antibiotics at zeptomole level based on target-aptamer binding triggered multiple recycling amplification

In the work, a signal-on electrochemical DNA sensor based on multiple amplification for ultrasensitive detection of antibiotics has been reported. In the presence of target, the ingeniously designed hairpin probe (HP1) is opened and the polymerase-assisted target recycling amplification is triggered, resulting in autonomous generation of secondary target. It is worth noting that the produced secondary target could not only hybridize with other HP1, but also displace the Helper from the electrode. Consequently, methylene blue labeled HP2 forms a “close” probe structure, and the increase of signal is monitored. The increasing current provides an ultrasensitive electrochemical detection for antibiotics down to 1.3fM. To our best knowledge, such work is the first report about multiple recycling amplification combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

Ultrasensitive detection and characterization of molecules with infrared plasmonic metamaterials.

Infrared vibrational spectroscopy is an effective technique which enables the direct probe of molecular fingerprints, and such detection can be further enhanced by the emerging engineered plasmonic metamaterials. Here we experimentally demonstrate ultrasensitive detection and characterization of polymer molecules based on an asymmetric infrared plasmonic metamaterial, and quantitatively analyze the molecule detection sensitivity and molecule-structure interactions. A sharp, non-radiative Fano resonance supported by the plasmonic metamaterial exhibits strongly enhanced near-field, and the resonance frequency is tailored to match the vibrational fingerprint of the target molecule. By utilizing the near-field nature of the plasmonic excitation, significantly enhanced absorption signal of molecules in the infrared spectroscopy are obtained, enabling ultrasensitive detection of only minute quantities of organic molecules. The enhancement of molecular absorption up to 105 fold is obtained, and sensitive detection of molecules at zeptomole levels (corresponding to a few tens of molecules within a unit cell) is achieved with high signal-to-noise ratio in our experiment. The demonstrated infrared plasmonic metamaterial sensing platform offers great potential for improving the specificity and sensitivity of label-free, biochemical detection.

Simultaneously sizing and quantitating zeptomole-level DNA at high throughput in free solution.

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare-narrow-capillary/hydrodynamic-chromatography or BaNC-HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC-HDC for measuring the sizes and quantities of DNA fragments at zmol to several-molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC-HDC can be a cost-effective means and an excellent tool for high-throughput DNA sizing and quantitation at extremely low quantity level.

Cooperative Amplification-Based Electrochemical Sensor for the Zeptomole Detection of Nucleic Acids

In this work, we developed a multiple-amplification-based electrochemical sensor for ultrasensitive detection of nucleic acids using a disease-related sequence of the p53 gene as the model target. A capture probe (CP) with a hairpin structure is immobilized on the electrode surface via thiol-gold bonding, while its stem is designed to contain a restriction site for EcoRI. In the absence of target DNA, the probe keeps a closed conformation and forms a cleavable region. After treatment with EcoRI, the target binding portion (loop) plus the biotin tag can be peeled off, suppressing the background current. In contrast, the CP is opened by the target hybridization, deforming the restriction site and forcing the biotin tag away from the electrode. On the basis of the biotin-streptavidin complexation, gold nanoparticles (GNPs) modified with a large number of ferrocene-signaling probes (Fc-SPs) are captured by the resulting interface, producing an amplified electrochemical signal due to the GNP-based enrichment of redox-active moieties. Furthermore, Fc tags can be dragged in close proximity to the electrode surface via hybridization between the signaling probes and the CP residues after EcoRI treatment, facilitating interfacial electron transfer and further enhancing the signal. With combination of these factors, the present system is demonstrated to achieve an ultrahigh sensitivity of zeptomole level and a wide dynamic response range of over 7 orders of magnitude.

Selective DNA detection at Zeptomole level based on coulometric measurement of gold nanoparticle-mediated electron transfer across a self-assembled monolayer

A selective DNA sensing with zeptomole detection level is developed based on coulometric measurement of gold nanoparticle (AuNPs)-mediated electron transfer (ET) across a self-assembled monolayer on the gold electrode. After immobilization of a thiolated hairpin-structured DNA probe, an alkanethiol monolayer was self-assembled on the resultant electrode to block [Fe(CN)6]3−/4− in a solution from accessing the electrode. In the presence of DNA target, hybridization between the DNA probe and the DNA target breaks the stem duplex of DNA probe. Consequently, stem moiety at the 3′-end of the DNA probes was removed from the electrode surface and made available for hybridization with the reporter DNA-AuNPs conjugates (reporter DNA-AuNPs). The thiolated reporter DNA matches the stem moiety at the 3′-end of the DNA probe. AuNPs were then enlarged by immersing the electrode in a growth solution containing HAuCl4 and H2O2 after the reporter DNA-AuNPs bound onto the electrode surface. The enlarged AuNPs on the electrode restored the ET between the electrode and the [Fe(CN)6]3−/4−, as a result, amplified signals were achieved for DNA target detection using the coulometric measurement of Fe(CN)6 3− electro-reduction by prolonging the electrolysis time. The quantities of ET on the DNA sensor increased with the increase in DNA target concentration through a linear range of 3.0 fM to 1.0 pM when electrolysis time was set to 300 s, and the detection limit was 1.0 fM. Correspondingly, thousands of DNA (zeptomole) copies were detected in 10-μL samples. Furthermore, the DNA sensor showed excellent differentiation ability for single-base mismatch.

Ultra-sensitive vibrational spectroscopy of protein monolayers with plasmonic nanoantenna arrays

Infrared absorption spectroscopy enabling direct access to vibrational fingerprints of the molecular structure is a powerful method for functional studies of bio-molecules. Although the intrinsic absorption cross-sections of IR active modes of proteins are nearly 10 orders of magnitude larger than the corresponding Raman cross-sections, they are still small compared to that of fluorescence-label based methods. Here, we developed a new tool based on collective excitation of plasmonic nanoantenna arrays and demonstrated direct detection of vibrational signatures of single protein monolayers. We first tailored the geometry of individual nanoantennas to form resonant structures that match the molecular vibrational modes. The tailored nanoantennas are then arranged in such a way that their in-phase dipolar coupling leads to a collective excitation of the ensemble with strongly enhanced near fields. The combined collective and individual plasmonic responses of the antenna array play a critical role in attaining signal enhancement factors of 10(4)-10(5). We achieved measurement of the vibrational spectra of proteins at zeptomole levels for the entire array, corresponding to only 145 molecules per antenna. The near-field nature of the plasmonic enhancement of the absorption signals is demonstrated with progressive loading of the nanoantennas with varying protein film thicknesses. Finally, an advanced model based on nonequilibrium Green's function formalism is introduced, which explains the observed Fano-type absorption line-shapes and tuning of the absorption strengths with the antenna resonance.

Dual‐asymmetry electrokinetic flow focusing for pre‐concentration and analysis of catecholamines in CE electrochemical nanochannels

In this research, a technique incorporating dual-asymmetry electrokinetic flow (DAEKF) was applied to a nanoCE electrochemical device for the pre-concentration and detection of catecholamines. The DAEKF was constructed by first generating a zeta-potential difference between the top and bottom walls, which had been pre-treated with O2 and H2O surface plasma, respectively, yielding a 2-D gradient shear flow across the channel depth. The shear flow was then exposed to a varying zeta-potential along the downstream direction by control of the field-effect in order to cause downward rotational flow in the channel. By this mechanism, almost all of the samples were effectively brought down to the electrode surface for analysis. Simulations were carried out to reveal the mechanism of concentration caused by the DAEKF, and the results reasonably describe our experiment findings. This DAEKF technique was applied to a glass/glass CE electrochemical nanochip for the analysis of catecholamines. The optimum detection limit was determined to be 1.25 and 3.3 nM of dopamine and catechol, respectively. A detection limit at the zeptomole level for dopamine can be obtained in this device, which is close to the level released by a single neuron cell in vitro.