Label-free Biosensor Research Articles

Signal Amplification in Electrochemical DNA Biosensors Using Target-Capturing DNA Origami Tiles

Electrochemical DNA (e-DNA) biosensors are feasible tools for disease monitoring, with their ability to translate hybridization events between a desired nucleic acid target and a functionalized transducer, into recordable electrical signals. Such an approach provides a powerful method of sample analysis, with a strong potential to generate a rapid time to result in response to low analyte concentrations. Here, we report a strategy for the amplification of electrochemical signals associated with DNA hybridization, by harnessing the programmability of the DNA origami method to construct a sandwich assay to boost charge transfer resistance (RCT) associated with target detection. This allowed for an improvement in the sensor limit of detection by two orders of magnitude compared to a conventional label-free e-DNA biosensor design and linearity for target concentrations between 10 pM and 1 nM without the requirement for probe labeling or enzymatic support. Additionally, this sensor design proved capable of achieving a high degree of strand selectivity in a challenging DNA-rich environment. This approach serves as a practical method for addressing strict sensitivity requirements necessary for a low-cost point-of-care device.

A label-free multicolor colorimetric and fluorescence dual mode biosensing of HIV-1 DNA based on the bifunctional NiFe2O4@UiO-66 nanozyme

Finding the DNA of the human immune deficiency virus (HIV) with simple and sensitive detection is the main challenge in early diagnosis of AIDS. Herein, two-point separation strategies based on the colorimetric and fluorescence are introduced. The naked-eye qualitative and semiquantitative colorimetric, and also accuracy fluorescence quantification of HIV-1 DNA were applied using label-free NiFe2O4@UiO-66 nanozyme with both functions of peroxidase-mimetic like and emitting fluorescence. The DNA probe-conjugated nanozyme is employed to hybridize a sequence of HIV-1. NiFe2O4@UiO-66 nanozymes catalyze the decomposition of H2O2 to •OH which can produce a remarkable fluorescent product 2-hydroxyterephthalic acid (TAOH) by the oxidation of the bridging ligand of weakly fluorescent terephthalic acid (TA). The accessibility of H2O2 toward confined-NiFe2O4 MNPs was reduced by increasing the HIV-1 target DNA concentration, resulting in the fluorescence intensity of TAOH being decreased. Meanwhile, remaining the unreacted H2O2 was transferred an acidic colorimetric solution containing FeSO4 and gold nanorods (AuNRs). Increasing the amount of H2O2 available for longitudinal etching of AuNRs due to •OH-generating Fe+2-catalyzed H2O2 is reponsible for different colors from brownish to colorless depending on the HIV-1 target DNA concentration. The fluorescence intensity and obtained colors have offered the sensitive biosensing methods with a linear range from 0.05 to 300 and 1–200 pM, respectively with a detection limit as low as 1 fM. Our study revealed that the applied sensing assay provides a cost-effective and straightforward qualitative, semiquantitative, and sensitive quantitation visible monitoring without the necessity of high-end instruments for HIV-1 detection in a human blood plasma/serum samples.

Adsorption of tetracycline antibiotics to gold nanoparticles and feasibility of aptamer-based label-free colorimetric detection

Tetracyclines are a group of very important antibiotics that are still in use. To extract, detect, and remove tetracyclines from the environment, various nanomaterials have been employed. Although gold nanoparticles (AuNPs) are a commonly cited material for these purposes, a fundamental understanding of these tetracycline-AuNP systems is still limited. In this work, the adsorption of tetracycline, oxytetracycline, and doxycycline to AuNPs was studied. The effect on the colloidal stability of AuNPs, adsorption kinetics, and the resulting adsorption isotherms was measured. While millimolar concentrations of the tetracyclines can cause aggregation of AuNPs, saturated monolayer adsorption was achieved with low micromolar concentrations of the tetracyclines. Adsorption was instantaneous, and adsorption to AuNPs enhanced their intrinsic fluorescence instead of quenching. With the assumption that aptamer/target complexes cannot be easily adsorbed by AuNPs compared to free aptamers, a label-free colorimetric detection method was tested. While the label-free sensor showed target-dependent aggregation of AuNPs, a nonbinding mutant aptamer showed the same trend, suggesting that the color change did not reflect aptamer adsorption but other events such as target adsorption. This study indicates the importance of the fundamental understanding of target/AuNP interactions to correctly design aptamer and AuNP-based label-free biosensors.

Ultra-sensitive label-free SERS biosensor with high-throughput screened DNA aptamer for universal detection of SARS-CoV-2 variants from clinical samples

COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10−18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.

Label-free electrochemical DNA biosensing of MR TV 29 18s ribosomal RNA gene of Trichomonas vaginalis by signalization of non-spherical gold nanoparticles

Label-free electrochemical DNA biosensing of MR TV 29 18s ribosomal RNA gene of Trichomonas vaginalis by signalization of non-spherical gold nanoparticles

Bacteriophage-based nano-biosensors for the fast impedimetric determination of pathogens in food samples

The early and rapid detection of pathogenic microorganisms is of critical importance in addressing serious public health issues. Here, a new bacteriophage-based nano-biosensor was constructed and the electrochemical impedimetric method was fully optimized and applied for the quantitative detection of Escherichia coli O157:H7 in food samples. The impact of using a nanocomposite consisting of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs), and tungsten oxide nanostructures (WO3) on the electrochemical performance of disposable screen printed electrodes was identified using the cyclic voltammetry and electrochemical impedance spectroscopy. The use nanomaterials enabled high capturing sensitivity against the targeting bacterial host cells with the limit of detection of 3.0 CFU/ml. Moreover, selectivity of the covalently immobilized active phage was tested against several non-targeting bacterial strains, where a high specificity was achieved. Thus, the targeting foodborne pathogen was successfully detected in food samples with high specificity, and the sensor provided an excellent recovery rate ranging from 90.0 to 108%. Accordingly, the newly developed phage-biosensor is recommended as a disposable label-free impedimetric biosensor for the quick and real-time monitoring of food quality.

Enhanced Photoelectrochemical Detection of Alpha-Fetoprotein Using Dipole Image Effect and Lock-In Amplification

Alpha-fetoprotein (AFP) is a serum biomarker mainly for early stage diagnosis and prognosis of primary liver cancer in clinical analysis. As a label-free analyte-specific biosensor with ultrahigh sensitivity and broad detection range, photoelectrochemical (PEC) sensor is promising for AFP detection. A unique feature of the PEC sensor, the separation of its excitation source and the detection signal, offers a great opportunity in the spatial and temporal domains to enhance its performance. For the spatial domain, AuNPs/TiO2/Au (in short, ATA) sandwiched structure was designed for magnetic resonance generation and light absorption enhancement. The strong light–matter interaction due to the dipole image effect has achieved an over 20 times increase in the sensitivity of the designed sensor. Instead of the typical method that manually switches the light source for photocurrent, lock-in amplification (LIA) was proposed to modulate the light source in the temporal domain. Such modulated signal can be coherently amplified and extracted from a strong noise background, which improves the signal-to-noise ratio (SNR) of the photocurrent signal and leads to a larger measurement range. This designed AFP biosensor exhibited one-order-of-magnitude wider linear detection range from 10 pg mL <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{-{1}}$ </tex-math></inline-formula> to <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$1~\mu \text{g}$ </tex-math></inline-formula> mL <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{-{1}}$ </tex-math></inline-formula> with a detection limit of 4.5 pg mL <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{-{1}}$ </tex-math></inline-formula> .

Label-Free cancer cell biosensor based on photonic crystal ring resonator

A photonic crystal (PhC) biosensor is a compact device, which detects the changes of characteristic of the light due to binding the sample by the analyte. In this paper; we propose using ring resonator in two-dimensional (2D) photonic crystal biosensor. A periodic array of silicon rods with the refractive index of n = 3.46 in the air bacground formed a cubic lattice structure. The sensing mechanism occures when the the resonant wavelength shifts due to change in the the refractive index of analytes. The transmission spectrum shifts about 4.47 nm to the higher wavelengths by increasing the refractive index of analyte. This mechanism can recognize the normal, and cancerous cells. The average values of sensitivity, quality factor, FOM, and transmission for the designed structure are 308.5 (nm/RIU), 3803.55, 848.06 RIU-1 and 98.78 %, respectively. The reported structure has high quality promising applications for highly sensitive label-free optical biosensors.

Label-Free Electrochemical Biosensor Platforms for Cancer Diagnosis: Recent Achievements and Challenges.

With its fatal effects, cancer is still one of the most important diseases of today's world. The underlying fact behind this scenario is most probably due to its late diagnosis. That is why the necessity for the detection of different cancer types is obvious. Cancer studies including cancer diagnosis and therapy have been one of the most laborious tasks. Since its early detection significantly affects the following therapy steps, cancer diagnosis is very important. Despite researchers' best efforts, the accurate and rapid diagnosis of cancer is still challenging and difficult to investigate. It is known that electrochemical techniques have been successfully adapted into the cancer diagnosis field. Electrochemical sensor platforms that are brought together with the excellent selectivity of biosensing elements, such as nucleic acids, aptamers or antibodies, have put forth very successful outputs. One of the remarkable achievements of these biomolecule-attached sensors is their lack of need for additional labeling steps, which bring extra burdens such as interference effects or demanding modification protocols. In this review, we aim to outline label-free cancer diagnosis platforms that use electrochemical methods to acquire signals. The classification of the sensing platforms is generally presented according to their recognition element, and the most recent achievements by using these attractive sensing substrates are described in detail. In addition, the current challenges are discussed.

Exfoliated Flexible Photonic Crystal Slabs for Refractive Index and Biomolecular Sensing

Using the optical transduction properties of one-dimensional photonic crystal slabs is a promising approach for label-free biosensing in the field of flexible and wearable biosensing. In this work we demonstrate the fabrication of flexible photonic crystal slabs (f-PCS) via an exfoliation method from rigid photonic crystal slabs. We investigate the processing effect on the optical properties and show that the optical resonances are maintained, but affected. The quality factor changes from 50 to 31 for the TE mode and from 187 to 136 for the TM mode after the process. We demonstrate use of f-PCS for refractive index sensing and achieve a limit of detection (LOD) of 3.6 E-4 refractive index units (RIU). Furthermore, we introduce vapor-phase functionalization of the f-PCS and show first results of biosensing of antibodies from diluted feline serum.

Label-Free Electrochemical Methods for Disease Detection.

Label-free electrochemical biosensing leverages the advantages of label-free techniques, low cost, and fewer user steps, with the sensitivity and portability of electrochemical analysis. In this review, we identify four label-free electrochemical biosensing mechanisms: (a) blocking the electrode surface, (b) allowing greater access to the electrode surface, (c) changing the intercalation or electrostatic affinity of a redox probe to a biorecognition unit, and (d) modulating ion or electron transport properties due to conformational and surface charge changes. Each mechanism is described, recent advancements are summarized, and relative advantages and disadvantages of the techniques are discussed. Furthermore, two avenues for gaining further diagnostic information from label-free electrochemical biosensors, through multiplex analysis and incorporating machine learning, are examined.

High-sensitivity polarization-independent terahertz Taichi-like micro-ring sensors based on toroidal dipole resonance for concentration detection of Aβ protein

Abstract Terahertz (THz) metamaterial sensor is a newly-developing interdisciplinary technology, which combines the essential characteristics of THz spectroscopy and metamaterials, to obtain better sensitivity for trace detection of the different target analytes. Toroidal dipole resonances show great sensing potential due to their suppression of the radiative loss channel. Here, we found a high-quality planar toroidal dipole resonance in the breaking Chinese Taichi-like ring and then designed a novel polarization-independent terahertz toroidal sensor by combining four Taichi-like rings into a cycle unit. The sensor shows high-sensitivity sensing characteristics for the ultrathin analyte and refractive index. The optimized sensitivity of pure analytes under 4 μm coating thickness can numerically reach 258 GHz/RIU in the corresponding ∼1.345 THz frequency domain, which is much higher than that of previously reported sensors. We further fabricated experimentally the sensor and demonstrated its fascinating polarization-independent characteristics. Finally, it was successfully applied to the low-concentration detection (ranging from 0.0001 mg/mL to 10 mg/mL) of Aβ protein associated with Alzheimer’s disease. Our high-sensitivity polarization-independent THz toroidal dipole sensor would give access to rich applications in label-free biosensing.

Electroplating-based engineering of plasmonic nanorod metamaterials for biosensing applications

Sensing lower molecular weight in a diluted solution using a label-free biosensor is challenging and requires a miniaturized plasmonic structure, e.g. a vertical Au nanorod (AuNR) array-based metamaterials. The sensitivity of a sensor mainly depends on transducer properties and hence for instance, the AuNR array geometry requires optimization. Physical vapour deposition methods (e.g. sputtering and e-beam evaporation) require a vacuum environment to deposit Au, which is costly, time-consuming, and thickness-limited. On the other hand, chemical deposition, i.e. electroplating deposit higher thickness in less time and at lower cost, becomes an alternative method for Au deposition. In this work, we present a detailed optimization for the electroplating-based fabrication of these metamaterials. We find that slightly acidic (6.0 < pH < 7.0) gold sulfite solution supports immersion deposition, which should be minimized to avoid uncontrolled Au deposition. Immersion deposition leads to plate-like (for smaller radius AuNR) or capped-like, i.e. mushroom (for higher radius AuNR) structure formation. The electroplating time and DC supply are the tuning parameters that decide the geometry of the vertically aligned AuNR array in area-dependent electroplating deposition. This work will have implications for developing plasmonic metamaterial-based sensors.

ZnO Tetrapods for Label-Free Optical Biosensing: Physicochemical Characterization and Functionalization Strategies

In this study, we fabricated three different ZnO tetrapodal nanostructures (ZnO-Ts) by a combustion process and studied their physicochemical properties by different techniques to evaluate their potentiality for label-free biosensing purposes. Then, we explored the chemical reactivity of ZnO-Ts by quantifying the available functional hydroxyl groups (-OH) on the transducer surface necessary for biosensor development. The best ZnO-T sample was chemically modified and bioconjugated with biotin as a model bioprobe by a multi-step procedure based on silanization and carbodiimide chemistry. The results demonstrated that the ZnO-Ts could be easily and efficiently biomodified, and sensing experiments based on the streptavidin target detection confirmed these structures' suitability for biosensing applications.

Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection.

The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 µM. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging.

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Integrated label-free erbium-doped fiber laser biosensing system for detection of single cell Staphylococcus aureus

A critical challenge to realize ultra-high sensitivity with optical fiber interferometers for label free biosensing is to achieve high quality factors (Q-factor) in liquid. In this work a high Q-factor of 105, which significantly improves the detection resolution is described based on a structure of single mode -core-only -single mode fiber (SCS) with its multimode (or Mach-Zehnder) interference effect as a filter that is integrated into an erbium-doped fiber laser (EDFL) system for excitation. In the case study, the section of core-only fiber is functionalized with porcine immunoglobulin G (IgG) antibodies, which could selectively bind to bacterial pathogen of Staphylococcus aureus (S. aureus). The developed microfiber-based biosensing platform called SCS-based EDFL biosensors can effectively detect concentrations of S. aureus from 10 to 105 CFU/mL, with a responsivity of 0.426 nm wavelength shift in the measured spectrum for S. aureus concentration of 10 CFU/mL. The limit of detection (LoD) is estimated as 7.3 CFU/mL based on the measurement of S. aureus with minimum concentration of 10 CFU/mL. In addition, when a lower concentration of 1 CFU/mL is applied to the biosensor, a wavelength shift of 0.12 nm is observed in 10% of samples (1/10), indicating actual LoD of 1 CFU/mL for the proposed biosensor. Attributed to its good sensitivity, stability, reproducibility and specificity, the proposed EDFL based biosensing platform has great potentials for diagnostics.

Terahertz chiral enantiomers recognition via chiral selective bio-molecular film on anapole metasurface sensor in water environment

Terahertz (THz) label-free and real-time metasurface-based biosensors have attracted significant attention in the biological field, but it is still challenging to achieve the efficient distinction of isomers, especially for absorptive liquid samples. Here, we demonstrated a chiral selective bio-molecular film constructed on an anapole metasurface sensor and realized the chiral recognition of the amino acid enantiomers. The proposed sensor can provide an ultrahigh localized field with a strong THz reflected signal even in a water environment. We utilized it to detect different concentrations of l-Met and l-Phe, the highest detection sensitivity is 0.516 GHz∙ml/μmol, and the detection limit can reach nanomole-level. More importantly, the sensor surface is modified by bovine serum albumin (BSA) through amine coupling, whichhas different binding abilities to D- and l-amino acids. The binding process has been monitored in real time, and the binding rate of l-amino acids to the metasurface is always higher than that of d-amino acids. The maximum sensitivity for l-amino acids is 2.23 times the d-type enantiomer. Our results illustrate a new avenue for chiral recognition of chiral samples, and it is expected that the biomolecule-metasurface hybrid structure is promising for developing THz sensors for biomolecular detection.

High sensitivity biosensing scheme based on a GHz phononic crystal waveguide

We propose a high sensitivity biosensor based on a GHz phononic crystal (PnC) waveguide, and demonstrate its operation by numerical simulations. The geometry consists of a micron-scale freestanding PnC silica waveguide plate with embedded Au nanopillars for bioparticle attachment, the PnC plate lying between two groups of periodic metal strips for GHz Lamb-wave acoustic generation and detection with ultrashort light pulses. By precise choice of the waveguide defect width, this biosensor is designed to work using a single, isolated waveguide mode. We study the influence of the waveguide defect width on the acoustic dispersion and transmission of this mode. Bioparticle attachment is simulated by investigation of the Au nanopillar mass loading, and is shown to shift the waveguide transmission peak to lower frequencies. We thereby demonstrate femtogram detection, showing that our approach provides a new methodology for label-free ultra-sensitive biosensing.

A light-up fluorescence platform based DNA: RNA hybrid G-quadruplet for detecting single nucleotide variant of ctDNA and miRNA-21

The nucleic acid assay is an area of great concern in the diagnosis and treatment of breast cancer. Here, we developed a DNA: RNA hybrid G-quadruplet (HQ) detection platform based on strand displacement amplification (SDA) and Baby Spinach RNA aptamer for single nucleotide variant (SNV) of circulating tumor DNA (ctDNA) and miRNA-21. This was the first in vitro construction of HQ for the biosensor. It found that HQ had much stronger ability to switch on fluorescence of DFHBI-1T than Baby Spinach RNA alone. Taking advantage of the platform and the FspI enzyme with high specificity, the biosensor achieved ultra-sensitive detection of SNV of the ctDNA (PIK3CA H1047R gene) and miRNA-21. The light-up biosensor had high anti-interference ability in complex actual samples. Hence, the label-free biosensor provided a sensitive and accurate method for early diagnosis of breast cancer. Moreover, it opened a new application model for RNA aptamers.