Bioanalytical Platform Research Articles

(Invited) Biosensors and Bioanalytical Platforms for in Situ Monitoring of Bacterial Phenotypes

: Bacterial cells are found everywhere and can be both beneficial or harmful to our diverse ecosystem, from plants to animals and humans. As such, studying them to better understand their interactions with the surrounding environment is important from both fundamental and applied perspectives. The existing analytical methods for studying bacterial cells require bulky readout equipment and complex sample preparation, are cell-destructive, or involve excessive sample preparation (1–3) which make real-time and in situ analysis hardly possible. In addition, probing bacterial growth has been traditionally done using colony counting, which is an end-point method and takes at least 24 hours for results.In situ monitoring of bacterial phenotypes (e.g. motion, respiration, adhesion, virulence factors) and how they change in response to environmental perturbation will have a broad impact in food safety, microbial fuels, studying gut-brain interaction, and fighting antibiotic resistance. This talk presents our recent works on developing novel biosensing tools for in situ and non-destructive probing and monitoring of bacterial phenotypes – from electrochemical sensors for reagent-free monitoring of metabolic activity or measuring biofilm virulence factors, to impedimetric sensors to decipher the mechanism of action of new antibiotics or activation of osmoregulatory transporters (4–7), to machine learning-enabled dynamic laser speckle imaging for monitoring bacterial motion (8), and others.

Electrochemiluminescence Enhanced by the Synergetic Effect of Porphyrin and Multi‐walled Carbon Nanotubes for Uric Acid Detection

AbstractThe design and exploration of efficient, stable and environmentally compatible organic emitters for an electrochemiluminescence (ECL) sensor is a promising topic. Herein, a novel environmentally‐friendly luminophore, ZnBCBTP@MWCNTs, were fabricated via self‐assembly of porphyrin molecules (ZnBCBTP) onto multi‐walled carbon nanotubes (MWCNTs). The resulting luminophore ZnBCBTP@MWCNTs displayed not only the highly ECL property and but also the good accelerated electron mobility. Then, a label‐free ECL biosensor based ZnBCBTP@MWCNTs was constructed for the ultrasensitive detection of uric acid. Excitingly, this proposed ECL biosensor performed a good linear relationship in the range of 0–300 μM with a low detection limit of 1.4 μM, thus offering another reliable and feasible sensing platform for clinical bioanalysis with good selectivity, stability, and repeatability.

Ratiometric electrochemiluminescence biosensor based on Ir nanorods and CdS quantum dots for the detection of organophosphorus pesticides

The poor solubility and limited ECL emission efficiency of iridium (Ir) complexes in aqueous restricted their application in ECL field. This work prepared water-soluble Ir nanorods (Ir NRs) via carboxyl-functionalizing tris (2-phenylpyridine) iridium(Ⅲ) (Ir(ppy)3) using poly(styrene-co-maleicanhydride) (PSMA) and further developed a highly sensitive dual-signal nanoprobe with Ir NRs as anodic emitter and CdS quantum dots (QDs) as cathodic emitter. Ir [email protected] QDs served as the matrix to immobilize enzymes to construct an ECL ratio biosensor for detecting organophosphorus pesticides (OPs). The dual-functional H2O2 produced by enzymatic reaction had an opposite effect on two ECL signals, namely enhanced the cathodic signal and quenched the anodic signal. With the OPs, the activity of enzyme was inhibited and the generation of H2O2 was prevented, thus the opposite changes of two ECL signals were detected. The detection of ethyl paraoxon (EP) as target OPs was achieved with a linear range from 5.0 × 10−12 to 5.0 × 10−8 M and detection limit was 1.67 × 10−12 M. The excellent solubility and high ECL efficiency of Ir NRs in aqueous greatly expanded the application of Ir(Ⅲ) complexes. The opposite effect of H2O2 on two emissions of Ir [email protected] QDs provided a novel ratio ECL platform for bioanalysis.

Dynamic light scattering immunosensor based on metal-organic framework mediated gold growth strategy for the ultra-sensitive detection of alpha-fetoprotein

The ultrasensitive detection of disease-related biomarkers is critical to improving the early diagnosis, monitoring, and treatment of diseases. Herein, we reported a dynamic light scattering (DLS)-based immunosensor for the high-sensitivity detection of alpha-fetoprotein (AFP), an important biomarker in the early diagnosis of primary liver cancer. A metal–organic framework and gold nanoparticle composite ([email protected]) was designed as a low-background scattering signal probe. After its structure was decomposed in a weak alkaline solution, the numerous small-sized AuNPs (∼4 nm) were released from the MOF structure. Controllable gold in situ growth strategy without self-nucleation was then executed to increase the AuNP size. Owing to the double signal amplification of the burst release of numerous small-sized AuNPs from MOFs and the gold in situ growth for an enhanced AuNP size, the proposed DLS immunosensor was successfully applied for the ultrasensitive detection of AFP with the detection limit of 0.36 pg mL−1. The reliability of the proposed method was evaluated by analyzing 22 actual clinic serum samples, and the results were further verified by the chemiluminescence immunosensor. This work features [email protected] and the gold in situ growth amplified modulation of DLS-based signal transduction, which demonstrates the great potential as a novel bioanalytical platform for a myriad of purposes.

Cellular and mitochondrial dual-targeted nanoprobe with near-infrared emission for activatable tumor imaging and photodynamic therapy

A nanotheranostic system integrating activatable photoactivity and dual targeting modalities is significant for bioimaging and biomedicine. In this study, a dual-targeting and tumor microenvironment responsive nanoassembly for imaging of HAase and PDT of tumor was developed. The synthesized water-soluble cationic porphyrin act as both the mitochondrion targeting photosensitizer and crosslinking agent to trigger the formation of HA-Mito TPP nanoparticle via the electrostatic interaction with hyaluronic acid. The nanoassembly could be efficiently endocytosed into targeted tumor cells and activated both fluorescence signal and enhanced PDT effect by highly expressed HAase. In vitro experiments suggested that the reported assay had desirable selectivity and excellent anti-interference for HAase quantitative analysis. The live cell studies showed the HA-Mito TPP had obviously enhanced PDT efficiency and caused mitochondria damage. Therefore, the developed dual-targeting nanotheranostic system provide a promising platform for bioanalysis and imaging guided photodynamic therapy.

Versatile Microfluidic Platform for Automated Live-Cell Hyperspectral Imaging Applied to Cold Climate Cyanobacterial Biofilms.

Microfluidic bioanalytical platforms are driving discoveries from synthetic biology to the health sciences. In this work, we present a platform for in vivo live-cell imaging and automated species detection in mixed cyanobacterial biofilms from cold climate environments. Using a multimodal microscope with custom optics applied to a chip with six parallel growth channels, we monitored biofilm dynamics via continuous imaging at natural irradiance levels. Machine learning algorithms were applied to the collected hyperspectral images for automatic segmentation of mixed-species biofilms into individual species of cyanobacteria with similar filamentous morphology. The coupling of microfluidic technology with modern multimodal imaging and computer vision systems provides a versatile platform for the study of cause-and-effect scenarios of cyanobacterial biofilms, which are important elements of many ecosystems, including lakes and rivers of the polar regions.

Ultrafast bacterial cell lysis using a handheld corona treater and loop-mediated isothermal amplification for rapid detection of foodborne pathogens

Bacterial foodborne diseases have become a growing global health concern, and nucleic acid-based analysis is considered as a promising approach for rapid identification of foodborne pathogens. Accordingly, nucleic acid isolation becomes the prerequisite first step for subsequent bioanalysis. However, current nucleic acid extraction suffers from operation time delayed, amplification inhibited by chemicals, or bulky instruments required. Here, we presented an ultrafast bacterial cell lysis induced by corona discharge within 40 s and combined it with loop-mediated isothermal amplification (LAMP) for the first time to achieve rapid and convenient detection of foodborne pathogens. Firstly, the viability and morphological changes revealed that corona discharge could destabilize bacterial cell envelope and eliminate more than 90% of Salmonella enterica (S. enterica) or Staphylococcus aureus (S. aureus) within 40 s. More surprisingly, more DNA were released from 100-103 CFU of electrical lysed S. enterica than equivalent thermal lysed ones, manifesting advantages of corona discharge on shorter preparation time and greater yields of DNA. Furthermore, the advantages of this ultrafast bacterial cell lysis were leveraged when collaborating with colorimetric LAMP. After conducting colorimetric LAMP in a water bath at 63 °C for 1 h, we could visually detect released DNA from 100 CFU of electrical lysed S. enterica and 103 CFU of electrical lysed S. aureus, respectively. Finally, we validated the applicability of the electrical lysis combined with LAMP reaction to detect (viable) S. enterica and S. aureus in artificially spiked pork samples. Therefore, we provided a rapid and convenient bioanalysis platform integrating an ultrafast electrical lysis and LAMP detection, opening up its extensive applications for versatile pathogens in resource-limited settings.

Target-Dependent Gating of Nanopores Integrated with H-Cell: Toward A General Platform for Photoelectrochemical Bioanalysis.

Herein we present a proof-of-concept study of target-dependent gating of nanopores for general photoelectrochemical (PEC) bioanalysis in an H-cell. The model system was constructed upon a left chamber containing ascorbic acid (AA), the antibody modified porous anodic alumina (AAO) membrane separator, and a right chamber placed with the three-electrode system. The sandwich immunocomplexation and the associated enzymatic generation of biocatalytic precipitation (BCP) in the AAO nanopores would regulate the diffusion of AA from the left cell to the right cell, leading to a varied photocurrent response of the ZnInS nanoflakes photoelectrode. Exemplified by fatty-acid-banding protein (FABP) as the target, the as-developed protocol achieved good performance in terms of sensitivity, selectivity, reproducibility, as well as efficient reutilization of the working electrode. On the basis of an H-cell, this work features a new protocol of target-dependent gating-based PEC bioanalysis, which can serve as a general PEC analytical platform for various other targets of interest.

High-Throughput Methods in the Discovery and Study of Biomaterials and Materiobiology.

The complex interaction of cells with biomaterials (i.e., materiobiology) plays an increasingly pivotal role in the development of novel implants, biomedical devices, and tissue engineering scaffolds to treat diseases, aid in the restoration of bodily functions, construct healthy tissues, or regenerate diseased ones. However, the conventional approaches are incapable of screening the huge amount of potential material parameter combinations to identify the optimal cell responses and involve a combination of serendipity and many series of trial-and-error experiments. For advanced tissue engineering and regenerative medicine, highly efficient and complex bioanalysis platforms are expected to explore the complex interaction of cells with biomaterials using combinatorial approaches that offer desired complex microenvironments during healing, development, and homeostasis. In this review, we first introduce materiobiology and its high-throughput screening (HTS). Then we present an in-depth of the recent progress of 2D/3D HTS platforms (i.e., gradient and microarray) in the principle, preparation, screening for materiobiology, and combination with other advanced technologies. The Compendium for Biomaterial Transcriptomics and high content imaging, computational simulations, and their translation toward commercial and clinical uses are highlighted. In the final section, current challenges and future perspectives are discussed. High-throughput experimentation within the field of materiobiology enables the elucidation of the relationships between biomaterial properties and biological behavior and thereby serves as a potential tool for accelerating the development of high-performance biomaterials.

Magnetic propulsion of colloidal microrollers controlled by electrically modulated friction.

Precise control over the motion of magnetically responsive particles in fluidic chambers is important for probing and manipulating tasks in prospective microrobotic and bio-analytical platforms. We have previously exploited such colloids as shuttles for the microscale manipulation of objects. Here, we study the rolling motion of magnetically driven Janus colloids on solid substrates under the influence of an orthogonal external electric field. Electrically induced attractive interactions were used to tune the load on the Janus colloid and thereby the friction with the underlying substrate, leading to control over the forward velocity of the particle. Our experimental data suggest that the frictional coupling required to achieve translation, transitions from a hydrodynamic regime to one of mixed contact coupling with increasing load force. Based on this insight, we show that our colloidal microrobots can probe the local friction coefficient of various solid surfaces, which makes them potentially useful as tribological microsensors. Lastly, we precisely manipulate porous cargos using our colloidal rollers, a feat that holds promise for bio-analytical applications.

Ultrasensitive fluorometric biosensor based on Ti3C2 MXenes with Hg2+-triggered exonuclease III-assisted recycling amplification.

Herein a rapid and sensitive fluorometric bioanalysis platform for mercury(ii) (Hg2+) detection was innovatively developed using ultrathin two-dimensional MXenes (Ti3C2) as fluorescence quencher and Hg2+-induced exonuclease III (Exo III)-assisted target recycling strategy for efficient signal amplification. Initially, fluorophore-labeled single-stranded DNA (FAM-labeled probe) can be easily adsorbed onto the surface of ultrathin Ti3C2 nanosheets by hydrogen bonding and metal chelating interaction, and the fluorescence signal emitted by the FAM-labeled probe is quenched strongly owing to the fluorescence resonance energy transfer between the FAM and ultrathin Ti3C2 nanosheets. Upon sensing the target Hg2+, the protruding DNA fragment at the 3' end of hairpin will hybridize with primer (hairpin-Hg2+-primer), and then further digested by Exo III to produce a probe (nicker). The released target Hg2+ and primer continue to participate in the next recycling, resulting in more hairpin probes becoming nickers. The combination of a large number of nickers and FAM-probe resulted in a significant increase in the fluorescence signal of the system, which was attributed to the fact that the double helix DNA was more rigid and separated from the surface of the ultrathin Ti3C2 nanosheets. The obvious fluorescence signal change of the Ti3C2-based Exo III-assisted target recycling can be accurately monitored by fluorescence spectrometry, which is also proportional to the concentration of Hg2+. Under optimum operating conditions, the peak intensity (520 nm wavelength) of fluorescence increased with increasing Hg2+ within a wide dynamic working range from 0.05 nM to 50 nM (R2 = 0.9913) with a limit of detection down to 42.5 pM. The proposed strategy uses ultrathin MXenes as a platform for binding nucleic acids, which contributes to its potential in nucleic acid hybridization-based biosensing and/or nucleic acid signal amplification bio-applications.

On-line coupling of two-phase microelectroextraction to capillary electrophoresis – Mass spectrometry for metabolomics analyses

We have integrated two-phase electroextraction (EE), capillary electrophoresis (CE) and mass spectrometry (MS) to combine rapid sample extraction, high performance separation and sensitive detection of metabolites. EE took place from a sample vial containing organic donor phase into a ~100 nL droplet of background electrolyte, which was hanging at the inlet of the capillary. In order to enable the EE process while the outlet of the capillary was connected to the MS several modifications were made. A modification was made to the sheath-liquid CE-MS interface, based on the use of a corrosion-resistant titanium ESI sprayer and a 6-port valve to switch between the CE-MS sheath liquid and the EE make-up liquid. Furthermore, a counter-pressure was applied, in order to prevent EOF from retracting the droplet during EE.Then, using five model metabolites (namely, leucine, isoleucine, adenosine, phenylalanine and guanosine) and crystal violet (CV) the extraction time and voltage were optimized and found to be 2000 s and 1 kV, respectively. Using these optimized conditions, the effect of various sodium chloride concentrations was examined to assess the influence of varying salt concentrations in biological samples. A set of 9 amino acids was used to validate the method. The detection limits ranged between 5 and 100 nM. LODs were improved 50–250 times in comparison with conventional CE-MS. Finally, to demonstrate the potential of the EE-CE-MS platform for bioanalysis of volume-limited samples, a urine sample of 300 nL was analyzed. This resulted in detection of 122 putative metabolites. The results indicate that EE-CE-MS could become a powerful tool for metabolomics analyses of volume-limited samples.

3D NiO nanoflakes/carbon fiber meshwork: Facile preparation and utilization as general platform for photocathodic bioanalysis

Herein, we describe a customized approach for facile preparation of three-dimensional (3D) NiO nanoflakes (NFs)/carbon fiber meshwork (CFM) and its validation as a common photocathode matrix for photoelectrochemical (PEC) bioanalysis, which to our knowledge has not been reported. Specifically, 3D NiO NFs/CFM was fabricated by a sequential liquid phase deposition and annealing process, which was then characterized by scanning electron microscopy, X–ray photoelectron spectrum, UV–vis absorption spectra and N2 adsorption-desorption measurement. Sensitized by BiOI and incorporated with an alkaline phosphatase (ALP)/tyrosinase (TYR) bi-enzyme cascade system, a sensitive split-type cathodic PEC bioanalysis for the determination of ALP was achieved. This method can detect ALP concentrations down to 3 × 10−5 U L−1 with a linear response range of 0.001–10 U L−1. Moreover, this proposed system exhibited good selectivity, stability and excellent performance for real sample analysis. This research features the facile preparation of 3D NiO NFs/CFM that could acts as a universal matrix for photocathodic analysis, and is envisioned to stimulate more effort for advanced 3D photocathode for PEC bioanalysis and beyond.

Fabrication of Infrared-Compatible Nanofluidic Devices for Plasmon-Enhanced Infrared Absorption Spectroscopy

Nanofluidic devices have offered us fascinating analytical platforms for chemical and bioanalysis by exploiting unique properties of liquids and molecules confined in nanospaces. The increasing interests in nanofluidic analytical devices have triggered the development of new robust and sensitive detection techniques, especially label-free ones. IR absorption spectroscopy is one of the most powerful biochemical analysis methods for identification and quantitative measurement of chemical species in the label-free and non-invasive fashion. However, the low sensitivity and the difficulties in fabrication of IR-compatible nanofluidic devices are major obstacles that restrict the applications of IR spectroscopy in nanofluidics. Here, we realized the bonding of CaF2 and SiO2 at room temperature and demonstrated an IR-compatible nanofluidic device that allowed the IR spectroscopy in a wide range of mid-IR regime. We also performed the integration of metal-insulator-metal perfect absorber metamaterials into nanofluidic devices for plasmon-enhanced infrared absorption spectroscopy with ultrahigh sensitivity. This study also shows a proof-of-concept of the multi-band absorber by combining different types of nanostructures. The results indicate the potential of implementing metamaterials in tracking several characteristic molecular vibrational modes simultaneously, making it possible to identify molecular species in mixture or complex biological entities.

Aptamer-Functionalized Microdevices for Bioanalysis.

Aptamers have drawn great attention in the field of biological research and disease diagnosis for the remarkable advantages as recognition elements. They show unique superiority for facile selection, desirable thermal stability, flexible engineering, and low immunogenicity, complementing the use of conventional antibodies. Aptamer-functionalized microdevices offer promising properties for bioanalysis applications because of the compact sizes, minimal reaction volume, high throughput, operational feasibility, and controlled preciseness. In this review, we first introduce the innovative technologies in the selection of aptamers with microdevices and then highlight some advanced applications of aptamer-functionalized microdevices in bioanalysis field for diverse targets. Aptamer-functionalized microfluidic devices, microarrays, and paper-based and other interface-based microdevices are all bioanalysis platforms with huge potential in the near future. Finally, the major challenges of these microdevices applied in bioanalysis are discussed and future perspectives are also envisioned.

Non-amplified impedimetric genosensor for quantification of miRNA-21 based on the use of reduced graphene oxide modified with chitosan

We report here an impedimetric genosensor for the quantification of microRNA-21 using [Fe(CN)6]3−/4− as redox probe to transduce the hybridization event. The biosensing platform was built at a thiolated-gold electrode by covalent bond of reduced graphene oxide (RGO) modified with chitosan (CHIT) and further covalent attachment of the aminated DNA probe. GO was used to provide the carboxylic groups for the covalent attachment of CHIT and, once reduced, to improve the electroactivity of the resulting platform, while CHIT served as a bridge between the thiol and the aminated probe DNA. The proposed bioanalytical platform allows the label-free, non-amplified, simple and fast biosensing of microRNA-21, with a linear range between 1.0 × 10−12 M and 1.0 × 10−8 M, a sensitivity of (134 ± 4) ΩM−1 (r2 = 0.996), a detection limit of 300 fM, and a reproducibility of 5.9% for 1.0 × 10−12 M miRNA-21 and 2.2% for 1.0 × 10−9 M miRNA-21. The genosensor was successfully used for the quantification of microRNA-21 in enriched human blood serum, urine and saliva samples.

An Integrated Multi-Function Heterogeneous Biochemical Circuit for High-Resolution Electrochemistry-Based Genetic Analysis.

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.

3D-bioprinted all-inclusive bioanalytical platforms for cell studies

Innovative drug screening platforms should improve the discovery of novel and personalized cancer treatment. Common models such as animals and 2D cell cultures lack the proper recapitulation of organ structure and environment. Thus, a new generation of platforms must consist of cell models that accurately mimic the cells’ microenvironment, along with flexibly prototyped cell handling structures that represent the human environment. Here, we adapted the 3D-bioprinting technology to develop multiple all-inclusive high throughputs and customized organ-on-a-chip-like platforms along with printed 3D-cell structures. Such platforms are potentially capable of performing 3D cell model analysis and cell-therapeutic response studies. We illustrated spherical and rectangular geometries of bio-printed 3D human colon cancer cell constructs. We also demonstrated the utility of directly 3D-bioprinting and rapidly prototyping of PDMS-based microfluidic cell handling arrays in different geometries. Besides, we successfully monitored the post-viability of the 3D-cell constructs for seven days. Furthermore, to mimic the human environment more closely, we integrated a 3D-bioprinted perfused drug screening microfluidics platform. Platform’s channels subject cell constructs to physiological fluid flow, while its concave well array hold and perfused 3D-cell constructs. The bio-applicability of PDMS-based arrays was also demonstrated by performing cancer cell-therapeutic response studies.

A programmable epidermal microfluidic valving system for wearable biofluid management and contextual biomarker analysis

Active biofluid management is central to the realization of wearable bioanalytical platforms that are poised to autonomously provide frequent, real-time, and accurate measures of biomarkers in epidermally-retrievable biofluids (e.g., sweat). Accordingly, here, a programmable epidermal microfluidic valving system is devised, which is capable of biofluid sampling, routing, and compartmentalization for biomarker analysis. At its core, the system is a network of individually-addressable microheater-controlled thermo-responsive hydrogel valves, augmented with a pressure regulation mechanism to accommodate pressure built-up, when interfacing sweat glands. The active biofluid control achieved by this system is harnessed to create unprecedented wearable bioanalytical capabilities at both the sensor level (decoupling the confounding influence of flow rate variability on sensor response) and the system level (facilitating context-based sensor selection/protection). Through integration with a wireless flexible printed circuit board and seamless bilateral communication with consumer electronics (e.g., smartwatch), contextually-relevant (scheduled/on-demand) on-body biomarker data acquisition/display was achieved.

Three-dimensional spatiotemporal tracking of nano-objects diffusing in water-filled optofluidic microstructured fiber

Abstract Three-dimensional (3D) tracking of nano-objects represents a novel pathway for understanding dynamic nanoscale processes within bioanalytics and life science. Here we demonstrate 3D tracking of diffusing 100 nm gold nanosphere within a water-filled optofluidic fiber via elastic light scattering–based position retrieval. Specifically, the correlation between intensity and position inside a region of a fiber-integrated microchannel has been used to decode the axial position from the scattered intensity, while image processing–based tracking was used in the image plane. The 3D trajectory of a diffusing gold nanosphere has been experimentally determined, while the determined diameter analysis matches expectations. Beside key advantages such as homogenous light-line illumination, low-background scattering, long observation time, large number of frames, high temporal and spatial resolution and compatibility with standard microscope, the particular properties of operating with water defines a new bioanalytical platform that is highly relevant for medical and life science applications.