3D Porous Edible Scaffolds from Rye Secalin for Cell-Based Pork Fat Tissue Culturing.

Cellular agriculture holds hope for a sustainable alternative to conventional meat, yet multiple technical challenges remain. These include the large-scale production of edible scaffolds and culturing methods for fat tissues, which are key to meat texture, flavor, and nutritional values. Herein. we disclose our method in the facile fabrication of sponge-like plant protein scaffolds by applying commercial sugar cubes as highly permeable templates. The prepared secalin scaffolds feature a high porosity of 85-90%, fully interconnected pores, and high water stability. The mechanical properties of scaffolds could be tuned by varying sugar-to-protein weight ratio and post-water annealing treatment. Moreover, murine preadipocytes (3T3-L1) and porcine adipose-derived stem cells (ADSCs) readily infiltrate, adhere, proliferate, and differentiate on the secalin scaffolds to develop a fat tissue morphology. A cultured fat tissue was produced by culturing porcine ADSCs for 12 days, which remarkably resembles conventional porcine subcutaneous adipose tissue in appearance, texture, flavor, and fatty acid profiles. The research demonstrates the great potential of sponge-like secalin scaffolds for cultured fat tissue production.

Three-Dimensional Scaffolds for Intestinal Cell Culture: Fabrication, Utilization, and Prospects.

The intestine is a visceral organ that integrates absorption, metabolism, and immunity, which is vulnerable to external stimulus. Researchers in the fields such as food science, immunology, and pharmacology have committed to developing appropriate in vitro intestinal cell models to study the intestinal absorption and metabolism mechanisms of various nutrients and drugs, or pathogenesis of intestinal diseases. In the past three decades, the intestinal cell models have undergone a significant transformation from conventional two-dimensional cultures to three-dimensional (3D) systems, and the achievements of 3D cell culture have been greatly contributed by the fabrication of different scaffolds. In this review, we first introduce the developing trend of existing intestinal models. Then, four types of scaffolds, including Transwell, hydrogel, tubular scaffolds, and intestine-on-a-chip, are discussed for their 3D structure, composition, advantages, and limitations in the establishment of intestinal cell models. Excitingly, some of the in vitro intestinal cell models based on these scaffolds could successfully mimic the 3D structure, microenvironment, mechanical peristalsis, fluid system, signaling gradients, or other important aspects of the original human intestine. Furthermore, we discuss the potential applications of the intestinal cell models in drug screening, disease modeling, and even regenerative repair of intestinal tissues. This review presents an overview of state-of-the-art scaffold-based cell models within the context of intestines, and highlights their major advances and applications contributing to a better knowledge of intestinal diseases. Impact statement The intestine tract is crucial in the absorption and metabolism of nutrients and drugs, as well as immune responses against external pathogens or antigens in a complex microenvironment. The appropriate experimental cell model in vitro is needed for in-depth studies of intestines, due to the limitation of animal models in dynamic control and real-time assessment of key intestinal physiological and pathological processes, as well as the "R" principles in laboratory animal experiments. Three-dimensional (3D) scaffold-based cell cultivation has become a developing tendency because of the superior cell proliferation and differentiation and more physiologically relevant environment supported by the customized 3D scaffolds. In this review, we summarize four types of up-to-date 3D cell culture scaffolds fabricated by various materials and techniques for a better recapitulation of some essential physiological and functional characteristics of original intestines compared to conventional cell models. These emerging 3D intestinal models have shown promising results in not only evaluating the pharmacokinetic characteristics, security, and effectiveness of drugs, but also studying the pathological mechanisms of intestinal diseases at cellular and molecular levels. Importantly, the weakness of the representative 3D models for intestines is also discussed.

3D-Printed Prolamin Scaffolds for Cell-Based Meat Culture.

Cultivating meat from muscle stem cells in vitro requires 3D edible scaffolds as the supporting matrix. Electrohydrodynamic (EHD) printing is an emerging 3D-printing technology for fabricating ultrafine fibrous scaffolds with high precision microstructures for biomedical applications. However, edible EHD-printed scaffolds remain scarce in cultured meat (CM) production partly due to special requirements with regard to the printability of ink. Here, hordein or secalin is mixed, which are cereal prolamins extracted from barley or rye, with zein to produce pure prolamin-based inks, which exhibit favorable printability similar to common polycaprolactone ink. Zein/hordein and zein/secalin scaffolds with highly ordered tessellated structures are successfully fabricated after optimizing printing conditions. The prolamin scaffolds demonstrated good water stability and in vitro degradability due to the porous fiber surface, which is spontaneously generated by culturing muscle cells for 1 week. Moreover, mouse skeletal myoblasts (C2C12) and porcine skeletal muscle satellite cells (PSCs) can adhere and proliferate on the fibrous matrix, and a CM slice is produced by culturing PSCs on prolamin scaffolds with high tissue similarity. The upregulation of myogenic proteins shows that the differentiation process is triggered in the 3D culture, demonstrating the great potential of prolamin scaffolds in CM production.

A surface plasmon resonance enhanced photoelectrochemical immunoassay based on perovskite metal oxide@gold nanoparticle heterostructures.

An innovative visible light-driven photoelectrochemical (PEC) immunosensing system was reasonably established for the sensitive detection of prostate-specific antigen (PSA) by using perovskite metal oxide@gold nanoparticle heterostructures (BaTiO3/Au) as the photoactive materials. When plasmonic Au nanoparticles were directly decorated on BaTiO3, a several times surface plasmon resonance (SPR) enhancement of photocurrent density was induced via the injection of hot electrons from visible light-excited Au nanoparticles into the conduction band of BaTiO3, and the combination of BaTiO3 and Au nanoparticles was employed as a promising platform for developing a photoelectrochemical bioanalysis. As a proof of concept, PSA had been detected by the BaTiO3/Au nanocomposite-based PEC sensor. To design such an immunoassay protocol, a monoclonal anti-PSA capture antibody (cAb)-coated microplate and glucose oxidase/polyclonal anti-PSA detection antibody-modified gold nanoparticles (GOx-Au NP-dAb) were used as the immunoreaction platform and signal probe, respectively. Upon the addition of target PSA, a sandwiched immunocomplex was formed accompanying the immuno-recognition between the antigen and antibody, and then the carried GOx could oxidize glucose to produce H2O2. The photocurrent of the BaTiO3/Au nanocomposite-functionalized electrode amplified with increasing H2O2 concentration since H2O2 is considered as a good hole scavenger. On the basis of the above-mentioned mechanisms and the optimized conditions, the assembled PEC immunosensor was linear with the logarithm of the PSA concentration in the range of 0.01-40 ng mL-1 with a detection limit of 4.2 pg mL-1. It afforded rapid response, good precision, and high stability and specificity, implying its great promise in photoelectrochemical immunoassays. More generally, this system sets up an ideal PEC immunosensing system based on the BaTiO3/Au nanocomposites and represents an innovative and low-cost "signal-on" assay scheme for the practical quantitative screening of low-abundance proteins.

Photoelectrochemical immunoassay of aflatoxin B1 in foodstuff based on amorphous TiO2 and CsPbBr3 perovskite nanocrystals.

Aflatoxin B1 (AFB1) pollution is one of the most serious problems for food safety. In this paper, a split-type photoelectrochemical (PEC) immunoassay was designed for sensitive detection of AFB1 in foodstuffs by using amorphous TiO2 with all-inorganic perovskite CsPbBr3 nanocrystals (CsPbBr3/a-TiO2). The a-TiO2 layer not only improved the stability of CsPbBr3 nanocrystals, but also facilitated charge transfer, which resulted in the increasing photocurrent of the nanocomposites. Initially, a competitive-type enzyme immunoreaction was executed on a high-binding microplate between the analyte and alkaline phosphatase (ALP)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugate. Accompanied by the formation of the immunocomplex, the carried ALP triggered enzymatic hydrolysis to generate ascorbic acid (AA, as an electron donor) for increasing the photocurrent of the CsPbBr3/a-TiO2-modified electrode. Coupling with the competitive enzyme immunoassay, the photocurrent of the modified electrode decreased with the increase of target AFB1 concentration in a dynamic working range from 0.01 ng mL-1 to 15 ng mL-1 with a limit of detection (LOD) of 2.8 pg mL-1 under optimum conditions. Furthermore, the photoelectrochemical immunoassay was also utilized to detect AFB1 in peanut and corn samples, giving acceptable accuracy in comparison with the referenced AFB1 enzyme-linked immunosorbent assay (ELISA) method.

Photoelectrochemical bioanalysis of antibiotics on rGO-Bi2WO6-Au based on branched hybridization chain reaction.

Herein a versatile photoelectrochemical (PEC) bioanalysis platform for sensitive and specific screening of low-abundance antibiotics (kanamycin, Kana, used in this case) was innovatively designed using rGO-Bi2WO6-Au as photoactive matrix and target-induced branched hybridization chain reaction (t-bHCR) for efficient signal amplification. To realize the high-performance of our PEC bioanalysis system, rational introduction of reduced graphene oxide (rGO) and Au nanoparticles (Au NPs) greatly accelerated the electron transfer and enhances photoactivity. As expected, the ternary nanocomposite (i.e., rGO-Bi2WO6-Au) system with cascade energy level exhibited intense PEC signal responses thanks to multistep electron-transfer (MET) mechanism. Upon sensing the target Kana, t-bHCR is readily implemented, thus resulting in the assembly of numerous CuS nanoparticle (CuS NP). As a result, the loading CuS NPs from hyper-branched structure boosted the electron donors (ascorbic acid) consumption and enhanced the steric hindrance, synergistically decrease the photoelectric response. Under the optimized testing conditions, the t-bHCR-based PEC bioanalysis exhibited superior analytical performance with a linear range of 1 pM to 5 nM target Kana and limit of detection down to 0.78 pM. Additionally, favorable stability, great anti-interference ability and satisfactory accuracy for the analysis of actual samples were acquired. Impressively, the concept of t-bHCR-mediated provides an alternative to construct PEC bioanalysis and inspire more interest in the design of advanced PEC bioanalysis through nucleic acid-related signal amplification.

Branched Polyethylenimine-Modified Upconversion Nanohybrid-Mediated Photoelectrochemical Immunoassay with Synergistic Effect of Dual-Purpose Copper Ions.

This work developed a near-infrared (near-IR) light-activated non-enzymatic signal-off photoelectrochemical (PEC) immunoassay for the ultrasensitive detection of α-fetoprotein (AFP) on the basis of branched polyethylenimine (BPEI)-modified upconversion nanoparticle (UCNP)@CdTe quantum dot (QD) nanostructures by coupling with the synergistic effect of dual-purpose copper ions. Emission light originated from NaYF4:Yb,Er UCNP was directly utilized through the electrostatic bonding of CdTe QDs to excite the separation of electron-hole pairs, resulting in the generation of obvious photocurrent under a 980 nm laser. By using polyclonal antibody-labeled cupric oxide nanoparticle as the secondary antibody, the nanolabel was introduced into the monoclonal anti-AFP antibody-modified microplates in the presence of target AFP. After treatment with acid, the as-released copper ion decreased the photocurrent through the synergistic effect with two issues: one was initially to form coordination with BPEI on the surface of UCNP, and then the near-IR excitation light and upconversion luminescence were attenuated due to the internal filter effect; another was to snatch the electrons flowing from the valence band of CdTe QD as the exciton trapping sites. Under optimal conditions, the dual-purpose Cu2+-activated signal-off PEC immunosensing platform exhibited a dynamic linear range from 10 pg mL-1 to 50 ng mL-1, accompanying the decreasing photocurrent with the increment of AFP concentration at an experimental detection limit of 1.2 pg mL-1. Importantly, good accuracy was achieved by this method in comparison with the results with human AFP ELISA kit for analysis of human serum samples. This dual-purpose Cu2+-activated PEC immunoassay brings a promising, enzyme-free and innovative thinking for the detection of low-abundance biomarkers.

Etching reaction-based photoelectrochemical immunoassay of aflatoxin B1 in foodstuff using cobalt oxyhydroxide nanosheets-coating cadmium sulfide nanoparticles as the signal tags.

A new split-type photoelectrochemical (PEC) immunosensing platform was designed for sensitive detection of aflatoxin B1 (AFB1) in foodstuffs, coupling with enzymatic hydrolysate-triggered etching reaction of cobalt oxyhydroxide (CoOOH) on cadmium sulfide (CdS) nanoparticles-functionalized interface. Initially, the photosensitive electrode was prepared by coating CoOOH nanosheets on the surface of CdS nanoparticles to quench the photocurrent. Thereafter, a competitive-type enzyme immunoreaction was carried out on monoclonal anti-AFB1 antibody-conjugated magnetic bead by using alkaline phosphatase (ALP)-labeled bovine serum albumin-AFB1 (AFB1-BSA) conjugate as the competitor. With the formation of immunocomplex, the carried ALP hydrolyzed ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA) and phosphate. The former ascorbic acid produced etched or dissolved CoOOH nanosheets into Co2+ ions, thus resulting in the exposure of CdS nanoparticles on the surface to enhance the photocurrent of the modified electrode. Under optimum conditions, the photocurrent decreased linearly with the increasing AFB1 concentration in the dynamic range of 0.01-10 ng mL-1, and the limit of detection was 2.6 pg mL-1. The precision of this method (expressed as RSD) was ±8.6%. In addition, the accuracy was monitored by analyzing spiked food samples, and gave the well-matched results with the referenced ELISA method.

Palindromic Molecular Beacon Based Z-Scheme BiOCl-Au-CdS Photoelectrochemical Biodetection.

This work presented an innovative and rationally engineered palindromic molecular beacon (PMB) based "Z-scheme" photoelectrochemical (PEC) biosensing protocol for the selective screening of kanamycin (Kana) through DNA hybridization-induced conformational conversion. Interestingly, the ingeniously designed PMB integrated the multifunctional elements including recognition region, primer-like palindromic fragment, and polymerization-nicking template. The cosensitized structures consisted of CdS quantum dot functionalized hairpin DNA2 (QD-HP2) and region-selectively deposited gold nanoparticles onto {001} facets of bismuth oxychloride (BiOCl-Au). Compared with BiOCl-Au alone, the attachment of CdS QDs onto BiOCl-Au (i.e., BiOCl-Au-CdS QDs) exhibited evidently enhanced photocurrent intensity thanks to the synergistic effect of Z-scheme BiOCl-Au-CdS QDs. After incubation with target Kana, Kana-aptamer binding could induce the exposure of PMB region for hairpin DNA1 (HP1). The exposed palindromic tails hybridized with each other (like a molecular machine) to consume the substrates (dNTPs) and fuels (enzyme) for the releasing of numerous nick fragments (Nick). The as-generated nick fragments could specifically hybridize with the complementary region of QD-HP2, thus resulting in decreasing photocurrent because of the increasing spatial distance for electron transfer between two-type photosensitizers. Under optimum conditions, the PMB-based sensing system exhibited satisfying photocurrent responses toward target Kana within the working range from 50 to 5000 fM at a low detection limit of 29 fM. Impressively, the concept of a palindromic fragment-mediated primer-free biosensing strategy offers a new avenue for advanced development of efficient and convenient biodetection systems.

Ultrasensitive and label-free electrochemical aptasensor of kanamycin coupling with hybridization chain reaction and strand-displacement amplification.

This work reports the proof-of-concept of an ultrasensitive label-free electrochemical aptasensor for Kanamycin (Kana) detection coupling strand-displacement amplification (SDA) with hybridization chain reaction (HCR). In the presence of target Kana, the analyte triggers conformational change of hairpin HP1 (HP1) and two-staged SDA to produce short single-stranded DNA (S1) with the aid of KF polymerase and nicking endonuclease. Meanwhile, the as-produced S1 hybridizes with the immobilized hairpin HP2 (HP2) on the electrode to open the hairpin, thereby resulting in the formation of DNA duplex. Thereafter, DNA duplex is selectively digested by Exo III accompanying S1 recycling. The residual single-stranded probe (S2) on the electrode opens another two hairpins in sequence and propagates a chain reaction of hybridization events between two alternating hairpins (H1 and H2) to form a long nicked double-helix. Upon addition of redox-active methylene blue (MB), numerous indicators are intercalated into the grooves of double-helix DNA polymers, each of which produces an electrochemical signal within the applied potentials. Under optimum conditions, the SDA/HCR-based electrochemical aptasensor exhibits a high sensitivity for detection of Kana down to 36 fM with a linear range from 0.05 to 200 pM. Additionally, the as-prepared aptasensor is successfully employed to determinate the Kana in animal derived food (milk). With the advantages of high sensitivity, label-free strategy and excellent selectivity, the developed aptasensor possesses great potential application value in food-safety analysis field.

Near-Infrared Light-Excited Core-Core-Shell UCNP@Au@CdS Upconversion Nanospheres for Ultrasensitive Photoelectrochemical Enzyme Immunoassay.

A novel photoelectrochemical (PEC) enzyme immunoassay was designed for the ultrasensitive detection of alpha-fetoprotein (AFP) based on near-infrared (NIR) light-excited core-core-shell UCNP@Au@CdS upconversion nanospheres. Plasmonic gold (Au) between the sandwiched layers was not only utilized as an energy harvester for the collection of the incident light but also acted as an energy conveyor to transfer the energy from upconversion NaYF4:Yb3+,Er3+ (UCNP) to semiconductor CdS, thus exciting the efficient separation of electron-hole pairs by the generated H2O2 of enzyme immunoreaction under the irradiation of a 980 nm laser. By virtue of high catalytic activity of natural enzymes, gold nanoparticles heavily functionalized with glucose oxidase (GOx) and polyclonal anti-AFP antibody were utilized to generate H2O2. A sandwiched immunoreaction was first carried out in a monoclonal anti-AFP antibody-coated microplate by using an antibody-labeled gold nanoparticle as secondary antibody. Accompanying the gold nanoparticle, the carried GOx oxidized glucose in H2O2, thereby resulting in the enhanced photocurrent via capturing holes on the valence band of CdS to promote the separation of electron-hole pairs. Under optimum conditions, the NIR light-based PEC immunosensing system exhibited good photocurrent responses toward target AFP within the dynamic working range of 0.01-40 ng mL-1 at a detection limit of 5.3 pg mL-1. Moreover, the NIR light-based sensing platform had good reproducibility and high selectivity. Importantly, good well-matched results obtained from NIR light-based PEC immunoassay were acquired for the analysis of human serum specimens by using AFP ELISA kit as the reference.

A homogeneous electrochemical sensor for Hg2+ determination in environmental water based on the T-Hg2+-T structure and exonuclease III-assisted recycling amplification.

A simple, fast, sensitive, and homogeneous electrochemical sensor based on the T-Hg2+-T structure and exonuclease III-assisted recycling amplification has been constructed for mercury ion (Hg2+) detection. The cT and methylene blue-labeled DNA probes (MB-TDNA) were designed to contain poly T sequences, which were repulsed from the negatively charged indium tin oxide (ITO) electrode due to their abundant negative charges. Hg2+ could trigger the formation of double-stranded DNA (dsDNA) between two DNA probes owing to the stable T-Hg2+-T structure. Then, Exo III specifically recognizes the cleavage of the double-stranded structure to release a methylene blue-labeled mononucleotide fragment (MB-MF). Moreover, the release of the target Hg2+ induces new hybridization and produces a large number of MB-MFs; MB-MFs are not repulsed by the negatively charged ITO electrode surface, thus producing a significant current signal. Under optimal conditions, the differential pulse voltammetric (DPV) response had a linear relationship with the logarithm of Hg2+ concentration in the range of 1.0 nM-0.5 µM, and the proposed method displayed great applicability for detecting Hg2+ in tap-water samples.