Signal Amplification Platform Research Articles

Signal amplification platform based on 2D MOF-on-MOF architectures-derived Co-decorated carbon@nitrogen-doped porous carbon for enhanced electrochemical acetaminophen sensing

Two-dimensional (2D) carbon-carbon hybrids derived from metal-organic frameworks (MOFs) are regarded as an intriguing type of electrode material in electrochemical sensing. In this work, a Co-decorated carbon@nitrogen-doped porous carbon heterostructure (Co/C@NC) was prepared via the simple calcination of 2D ZIF-L(Co)@ZIF-8. In this MOF-on-MOF precursor, the outer ZIF-8 layer not only prevents the collapse of ZIF-L(Co) during calcination but also endows the outer carbon an extended surface area and porous structure for more accessible active sites and a fast mass transfer process. Meanwhile, the formed CoNPs could facilitate the generation of graphitic carbon layers, which enhances electrocatalytic activity and boosted conductivity. Owing to these merits, the Co/C@NC-based sensor displays high electrochemical activity for acetaminophen (APAP) detection with a wide linear range (4 × 10-7 - 2 × 10-4 M) and a lower detection limit (8.2 × 10-8 M). The constructed sensor has been utilized for the analysis of APAP in real samples, yielding acceptable recovery between 96.6% and 104.0%. This work presents an efficient and convenient method for designing MOF-on-MOF-derived 2D carbon-carbon hybrids, which hold a promising prospect in electrochemical analysis.

Enzyme cascade signal amplification platform with immobilized xanthine oxidase on nanozymes exhibiting enhanced POD-like activity for ultrasensitive dual-mode detection of xanthine

Xanthine (XA) metabolic disorders cause many diseases, hence early and precise XA detection is essential. Herein, a dual-mode enzyme cascade nanosensing platform based on Au/FMZIF-rGO/PCN nanomaterial with dual-emissive and high peroxidase-like (POD-like) activity was devised. The rapid electron transfer between Fe, Mn and P elements and the formation of ZIF-rGO/PCN hybrid enhanced POD-like activity. Aggregation-induced emission (AIE) effect improved fluorescence intensity of AuNCs. Furthermore, utilizing the nanozyme to immobilize xanthine oxidase (XOD) also reduced intermediate product diffusion, improving cascade efficiency and platform detection·H2O2 was catalytically decomposed to generate •OH, lowering fluorescence intensity at 627 nm and oxidizing TMB to produce blue oxTMB. So, dual-mode nanosensing platform was constructed with linear ranges for ratio fluorescence and colorimetric detection of XA spanning 0.5–200 μM and 0.5–300 μM, respectively, and corresponding LODs of 0.11 and 0.24 μM. The nanoplatform suggested promising potential for practical clinical detection of XA and other biomarkers.

Ultrasensitive and Highly Selective Detection of Staphylococcus aureus at the Single-Cell Level Using Bacteria-Imprinted Polymer and Vancomycin-Conjugated MnO2 Nanozyme.

Pathogenic bacterial infections, even at extremely low concentrations, pose significant threats to human health. However, the challenge persists in achieving high-sensitivity bacterial detection, particularly in complex samples. Herein, we present a novel sandwich-type electrochemical sensor utilizing bacteria-imprinted polymer (BIP) coupled with vancomycin-conjugated MnO2 nanozyme (Van@BSA-MnO2) for the ultrasensitive detection of pathogenic bacteria, exemplified by Staphylococcus aureus (S. aureus). The BIP, in situ prepared on the electrode surface, acts as a highly specific capture probe by replicating the surface features of S. aureus. Vancomycin (Van), known for its affinity to bacterial cell walls, is conjugated with a Bovine serum albumin (BSA)-templated MnO2 nanozyme through EDC/NHS chemistry. The resulting Van@BSA-MnO2 complex, serving as a detection probe, provides an efficient catalytic platform for signal amplification. Upon binding with the captured S. aureus, the Van@BSA-MnO2 complex catalyzes a substrate reaction, generating a current signal proportional to the target bacterial concentration. The sensor displays remarkable sensitivity, capable of detecting a single bacterial cell in a phosphate buffer solution. Even in complex milk matrices, it maintains outstanding performance, identifying S. aureus at concentrations as low as 10 CFU mL-1 without requiring intricate sample pretreatment. Moreover, the sensor demonstrates excellent selectivity, particularly in distinguishing target S. aureus from interfering bacteria of the same genus at concentrations 100-fold higher. This innovative method, employing entirely synthetic materials, provides a versatile and low-cost detection platform for Gram-positive bacteria. In comparison to existing nanozyme-based bacterial sensors with biological recognition materials, our assay offers distinct advantages, including enhanced sensitivity, ease of preparation, and cost-effectiveness, thereby holding significant promise for applications in food safety and environmental monitoring.

Immunosensing of carbohydrate antigen 19–9 based on covalent organic framework loaded Prussian blue as signal amplification platform

Immunosensing of carbohydrate antigen 19–9 (CA 19–9) by using covalent organic framework (COF) loaded Prussian blue (PB) as signal amplification platform was developed. Firstly, two-dimensional (2D) vinyl-rich COFTAGH-Dva (TAGH, triaminoguanidine hydrochloride; Dva, 1,4-benzenedicarboxaldehyd) was prepared to load PB, and further the Ab2 was assembled through the “click” reaction between –SH of Ab2 and vinyl of COFTAGH-Dva to construct the beacon complex of PB/COFTAGH-Dva/Ab2. Another 2D COFTp-BD (Tp, 2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde; BD, benzidine) rich in –NH- groups was combined with AuNPs to form AuNPs/COFTp-BD by in-situ reduction method, and then Ab1 was assembled through Au-S bond. In the process of electrochemical immunosensing, AuNPs/COFTp-BD was first assembled on glassy carbon electrode (GCE) to form AuNPs/COFTp-BD/GCE, which then adsorbed Ab1 by Au-S bond, and specifically and quantitatively captured CA 19–9 and the beacon complex of PB/COFTAGH-Dva/Ab2. With the increase of antigen CA 19–9, the redox peak signal of Fe2+/Fe3+ derived from PB in the beacon complex of PB/COFTAGH-Dva/Ab2 gradually increased. The linear range of the electrochemical immunosensor was 0.01–150 U/mL (U/mL represents the concentration of CA 19–9, which is the number of units of CA 19–9 per 1 mL volume) and the detection limit was 0.003 U/mL. This work provides a reference for constructing high-performance electrochemical immunosensor probes by using good biocompatibility and electroactive materials loaded on non electroactive COFs.

DNA framework signal amplification platform-based high-throughput systemic immune monitoring

Systemic immune monitoring is a crucial clinical tool for disease early diagnosis, prognosis and treatment planning by quantitative analysis of immune cells. However, conventional immune monitoring using flow cytometry faces huge challenges in large-scale sample testing, especially in mass health screenings, because of time-consuming, technical-sensitive and high-cost features. However, the lack of high-performance detection platforms hinders the development of high-throughput immune monitoring technology. To address this bottleneck, we constructed a generally applicable DNA framework signal amplification platform (DSAP) based on post-systematic evolution of ligands by exponential enrichment and DNA tetrahedral framework-structured probe design to achieve high-sensitive detection for diverse immune cells, including CD4+, CD8+ T-lymphocytes, and monocytes (down to 1/100 μl). Based on this advanced detection platform, we present a novel high-throughput immune-cell phenotyping system, DSAP, achieving 30-min one-step immune-cell phenotyping without cell washing and subset analysis and showing comparable accuracy with flow cytometry while significantly reducing detection time and cost. As a proof-of-concept, DSAP demonstrates excellent diagnostic accuracy in immunodeficiency staging for 107 HIV patients (AUC > 0.97) within 30 min, which can be applied in HIV infection monitoring and screening. Therefore, we initially introduced promising DSAP to achieve high-throughput immune monitoring and open robust routes for point-of-care device development.

Electrochemical peptide-based biosensor for the detection of the inflammatory disease biomarker, interleukin-1beta

This paper reports the development of a highly sensitive and selective electrochemical peptide-based biosensor for the detection of the inflammatory disease biomarker, interleukin-1beta (IL-1β). To this end, flower-like Au–Ag@MoS2-rGO nanocomposites were used as the signal amplification platform to achieve a label-free biosensor with a high sensitivity and selectivity. First, a high-affinity peptide for IL-1β was identified through biopanning with M13 random peptide libraries, and was newly designed by incorporating cysteine at the C-terminus. An IL-1β specific binding peptide was used as the bio-receptor, and the interaction between the IL-1β binding peptide and IL-1β was confirmed via enzyme-linked immunosorbent assay and various physicochemical and electrochemical analyses. Under optimal conditions, the biosensor achieved an ultrasensitive and specific IL-1β detection in a wide linear concentration range of 0–250 ng/mL with a picomolar-level detection limit (∼2.4 pM), low binding constant (∼0.62 pM), and a low coefficient of variation (<1.65 %). The biosensor was successfully utilized for IL-1β determination in the serum of Crohn's disease patients with a good correlation coefficient. In addition, the detection performance was comparable to that of commercially available IL-1β ELISA kit. This indicates that the electrochemical peptide-based biosensor may offer a potentially valuable platform for the clinical diagnosis of various inflammatory disease biomarkers.

Simultaneous Detection of Infectious Diseases Using Aptamer-Conjugated Gold Nanoparticles in the Lateral Flow Immunoassay-Based Signal Amplification Platform.

Various platforms for the accurate diagnosis of infectious diseases have been studied because of the emergence of coronavirus disease (COVID-19) in 2019. Recently, it has become difficult to distinguish viruses with similar symptoms due to the continuous mutation of viruses, and there is an increasing need for a diagnostic method to detect them simultaneously. Therefore, we developed a paper-based rapid antigen diagnostic test using DNA aptamers for the simultaneous detection of influenza A, influenza B, and COVID-19. Aptamers specific for each target viral antigen were selected and attached to AuNPs for application in a rapid antigen diagnosis kit using our company's heterogeneous sandwich-type aptamer screening method (H-SELEX). We confirmed that the three viruses could be detected on the same membrane without cross-reactivity based on the high stability, specificity, and binding affinity of the selected aptamers. Further, the limit of detection was 2.89 pg·mL-1 when applied to develop signal amplification technology; each virus antigen was detected successfully in diluted nasopharyngeal samples. We believe that the developed simultaneous diagnostic kit, based on such high accuracy, can distinguish various infectious diseases, thereby increasing the therapeutic effect and contributing to the clinical field.

A FokI-driven signal amplification platform for the simultaneous detection of multiple viral RNA pathogens

The combination of a nucleic acid amplification method through multiplexed RCA, coupled with signal amplification mediated by FokI-assisted digestion of dumbbell-like oligonucleotides, enhances the detection of multiple human respiratory viruses.

Dual signal amplification platform based on recombinant polymerase amplification and fluorescence-enhanced nanoparticles for sensitive detection of Salmonella

Salmonella is a common foodborne pathogen, and its detection is essential for managing public food safety. Lateral flow assay (LFA) strips are well suited for the purpose as they are inexpensive and easy to use. However, the sensitive detection of low-concentration targets requires the use of signal amplification strategies. This paper presents a LFA for the ultrasensitive detection of Salmonella based on recombinase polymerase amplification (RPA) and fluorescence-enhanced composite nanospheres dSiO2@CD@SiO2 (dCDS). RPA is capable of rapid (∼20 min) exponential amplification of bacterial DNA and does not require complex instrumentation. Mesoporous silica (dSiO2) has a large specific surface area and radial pore size, making it an excellent structural material for internal assembly. Nanoparticles obtained by loading carbon dots (CDs) had a strong fluorescence signal and stability. We used dCDSs as reporter molecules, which captured a large number of DNA-dCDS complexes on the test line of the LFA. With double signal amplification, the RPA and dCDS-based LFA showed greatly improved sensitivity: The limits of detection for Salmonella in buffer and milk were 3.8 CFU/mL and 36 CFU/mL, respectively. This study provides an inexpensive strategy for the sensitive detection of pathogenic bacteria.

An ultrasensitive unlabeled electrochemical immunosensor for the detection of cardiac troponin I based on Pt/Au–B,S,N-rGO as the signal amplification platform

In this study, an ultrasensitive unlabeled electrochemical immunosensor for the detection of cardiac troponin I (cTnI) was developed based on Pt/Au modified B,S,N co-doped reduced graphene oxide (Pt/Au–B,S,N-rGO) as a signal amplification platform. First-principles calculations were employed to analyze the electron density of states of Pt/Au–B,S,N-rGO, revealing an increase in the electron density of the graphene oxide (GO) states. Furthermore, scanning electron microscopy (SEM), X-ray photoelectron diffraction spectroscopy (XPS), and electrochemical detection were used to successfully construct and analyze Pt/Au–B,S,N-rGO. The results showed that B,S,N-rGO exhibited good electrochemical activity, and the Au/Pt NPs demonstrated excellent catalytic properties, which provided a strong foundation for achieving high-sensitivity detection. Moreover, the constructed unlabeled electrochemical immunosensor had an ideal linear range (0.1 pg/mL∼50 ng/mL) and detection limit (0.082 pg/mL). In human serum detection, the results of this immunosensor were essentially similar to the ELISA results for the same samples, which suggested that the immunosensor had a promising clinical application prospect for the detection of cTnI.

N-doped TiO2/Ti3C2-driven self-photocatalytic molecularly imprinted ECL sensor for sensitive and steady detection of dexamethasone

The conventional luminol-based electrochemiluminescence (ECL) biosensor suffers from hampered signal stability due to the self-decomposition of the H2O2 co-reactant. Here, we propose an N-doped TiO2/Ti3C2 heterojunction driven self-photocatalytic platform for ECL signal amplification and then combine it with molecular imprinting technology for sensitive and steady detection of dexamethasone (DXM). Unlike traditional cases involving specific catalysts or external electron injection, the initial luminescence of luminol in this new system is utilized as the excitation light of N-doped TiO2/Ti3C2 photocatalyst to promote the conversation of dissolved oxygen to H2O2, supplying more co-reactants to improve ECL of luminol in turn. Thanks to the heterojunction and self-photocatalytic cyclic amplification, this molecularly imprinted ECL sensor exhibits a wide linear range (1.0 × 10−6–1.0 × 101 μg mL−1) and a low detection limit, as well as excellent anti-interference capability, sensitivity, and stability. This work contributes to more reliable and steady detection of DXM and brings new insights into developing exogenous co-reactant-free self-enhancement ECL models for biosensor applications.

A signal-off photoelectrochemical sandwich-type immunosensor based on WO3/TiO2 Z-scheme heterojunction.

A sandwich "signal-off" type photoelectrochemical (PEC) immunosensor was fabricated based on a composite heterojunction of tungsten oxide/titanium oxide microspheres (WO3/TiO2) acting as signal amplification platform and carbon microspheres loaded by gold nanoparticles (Cs@Au NPs) utilized as the label for detecting antibody. WO3/TiO2 had excellent photoelectric performance, and the results of Mott-Schottky plots, open-circuit voltage, and electron spin resonance spectroscopy indicated that it belonged to the Z-scheme heterojunction transfer mechanism of photogenerated carriers. To achieve the sensitization of PEC immunosensor, Cs@Au NP-labeled immunocomplex can effectively reduce the photocurrent signal. The PEC immunosensors were fabricated under the optimal conditions of 1:1 WO3/TiO2 (molar ratio), 2.0mgmL-1 WO3/TiO2, and 1.5mgmL-1 Cs@Au NPs. Through comparison of the detection results of label-free and sandwich-type PEC immunosensors for nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we found that the sensitivity of the sandwich type was 2.53 times the label-free type, and the limit of detection was 0.006ngmL-1, i.e., 3.17 times lower than the label-free type. This demonstrates that the developed sandwich-type PEC immunosensor will have a brighter application prospect.

Multiple TMA-aided CRISPR/Cas13a platform for highly sensitive detection of IL-15 to predict immunotherapeutic response in nasopharyngeal carcinoma

BackgroundImmune checkpoint inhibitors (ICIs)-based treatments have been recommended as the first line for refractory recurrent and/or metastatic nasopharyngeal carcinoma (NPC) patients, yet responses vary, and predictive biomarkers are urgently needed....

Dual-Signal Cascaded Nucleic Acid Amplification Circuit-Loaded Metal-Organic Frameworks for Accurate and Robust Imaging of Intracellular MicroRNA.

Cascaded signal amplification technologies play an important role in the sensitive detection of lowly expressed biomarkers of interests yet are constrained by severe background interference and low cellular accessibility. Herein, we constructed a metal-organic framework-encapsulating dual-signal cascaded nucleic acid sensor for precise intracellular miRNA imaging. ZIF-8 nanoparticles load and deliver FAM-labeled upstream catalytic hairpin assembly (CHA) and Cy5-modified downstream hybridization chain reaction (HCR) hairpin reactants to tumor cells, enabling visualization of the target-initiated signal amplification process for double-insurance detection of analytes. The pH-responsive ZIF-8 nanoparticles effectively protect DNA hairpins from degradation and allow the release of them in the acid tumor microenvironment. Then, intracellular target miRNAs orderly trigger cascaded nucleic acid signal amplification reaction, of which the exact progress is investigated through the analysis of the fluorescence recovering process of FAM and Cy5. In addition, DNA@ZIF-8 nanoparticles improve measurement accuracy by dual-signal colocalization imaging, effectively avoiding nonspecific false-positive signals and enabling in situ imaging of miRNAs in living cells. A dual-signal colocalization strategy allows accurate target detection in living cells, and DNA@ZIF-8 provides a promising intracellular sensing platform for signal amplification and visual monitoring.

A label-free electrochemical immunosensor based on PtCoCu PNPs/NB-rGO as a dual signal amplification platform for sensitive detection of β-Amyloid1-42

In this work, a label-free electrochemical immunosensor based on popcorn-shaped PtCoCu nanoparticles supported on N- and B-codoped reduced graphene oxide (PtCoCu PNPs/NB-rGO) was constructed to sensitively detect concentration level of β-Amyloid1-42 oligomers (Aβ). The PtCoCu PNPs exhibits excellent catalytic ability due to its popcorn structure which improves the specific surface area and porosity, resulting in more active sites being exposed and fast transport paths for ion/electron. NB-rGO with large surface area and unique pleated structure could disperse PtCoCu PNPs through electrostatic adsorption and formation of d-p dative bonds between the metal ion and pyridinic N of NB-rGO. In addition, the doping of B atoms enhances the catalytic ability of GO enormously and achieves further signal amplification. Besides, both PtCoCu PNPs and NB-rGO are able to fix abundant antibodies through M(Pt, Co, Cu)-N bonds and amide bonds respectively without any other complex processing procedures such as carboxylation, ect. The designed platform achieved the dual amplification of electrocatalytic signal and effectively immobilization of antibodies. Under the optimum conditions, the designed electrochemical immunosensor presented wide linear rang (50.0 fg/mL ∼ 100 ng/mL) and low detection limits (3.5 fg/mL). The results demonstrated that the prepared immunosensor will be promising in sensitive detection of AD biomarkers.

Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells.

The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10-6 U μL-1. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.

Two-channel electrochemical immunosensor based on one-step-synthesized AuPt-boron-doped graphene electrode for CA153 detection

Herein, a novel dual-channel electrochemical immunosensor was fabricated via vertical growth of AuPt-decorated boron-doped graphene (AuPt-BG) nanosheets as a signal amplification platform to detect cancer antigen 153 (CA153). Highly open, porous AuPt-BG films were synthesized using one-step electron-assisted hot-filament chemical vapor deposition. The Au–Pt alloy nanoparticles were dispersed on BG nanosheets to improve their biocompatibility, and antibodies (Ab) were directly bonded to the AuPt-BG electrode. The architectures enlarged the loading of CA153Ab and efficiently catalyzed the Fe(CN)63–/4– reaction, ultimately amplifying the signals. This novel strategy allows the simultaneous detection of CA153 in the oxidation and reduction channels, improving the reliability of the detection results. The AuPt-BG-based immunosensor exhibited a lower detection limit (0.0012 mU mL−1, S/N = 3) and wider linear range (0.1–4 × 104 mU mL−1) along with improved reproducibility, selectivity, and stability for the assay of CA153. Owing to the high process controllability of AuPt-BG films, a large-area electrode for in-vitro analyses and a flexible microelectrode for in-vivo analyses were prepared, which confirmed that the AuPt-BG-based sensor is an ideal CA153 detection platform for clinical diagnosis and practical applications.

Cobalt nanoparticles decorated bamboo-like N-doped carbon nanotube as nanozyme sensor for efficient biosensing

• Bamboo-like N doped carbon nanotubes encapsulated with cobalt nanoparticles (Co-BNCNTs) was designed by one-step pyrolysis. • The bamboo-like structure could prevent the self-aggregation of the CoNPs, improve the stability of the CoNPs, and enhance the catalytic activity of the CoNPs. • The hollow tubular structure could shorten the distance for the reactants to reach the electrode surface and accelerate the mass transfer process. In this work, Co nanoparticles decorated bamboo-like N-doped carbon nanotubes (Co-BNCNTs) were design, which exhibited excellent oxidase-mimicking activity and could be served as a novel signal amplification platform for dopamine (DA) sensing. Co nanoparticles (CoNPs) encapsulated in bamboo-like N-doped carbon nanotubes (BNCNTs) could prevent their self-aggregation and expose more active sites. Besides, the N-doping in carbon materials could adjust the charge density and increase active sites. Moreover, the synergistic effect between CoNPs and BNCNTs also could contribute to enhance catalytic activity. In a word, excellent electrical conductivity, large electrochemically active area, multiple catalytically active sites are beneficial for DA detection. The Co-BNCNTs/GCE biosensor showed good stability and selectivity, a wide detection range of 0.5-150 μM, and a lower detection limit of 0.0342 μM. The Co-BNCNTs sensor was employed to detect the DA in the actual sample, which displayed a potential application in clinical diagnosis. Furthermore, our study presented an effective way to the synthesis of metal nanoparticles encapsulated into CNTs catalysts as nanozymes for the application in biosensing.

Porous Carbon Nanospheres and Gold Nanocomposite-Based Electrochemical Aptasensor for the Detection of Streptomycin

A novel electrochemical aptasensor modified with the nanocomposite of porous carbon nanospheres and Au urchins as the signal amplification and immobility platforms for aptamer was successfully constructed for ultrasensitive and selective determination of streptomycin. The streptomycin aptamer was fixed on the surface of the nanocomposite via the strong Au–S bond between Au urchins and aptamer. The target binding-induced conformational change of aptamer resulted in signal attenuation, which was expressed as “ΔI = IBSA − Istreptomycin.” Based on the synergic signal amplification platform, the as-prepared aptamer-based sensor showed a wider linearity to streptomycin from 0.01 to 350 ng/mL with a low detection limit of 5.0 pg/mL under the optimized condition. Finally, the aptasensor was operated in milk and honey to detect streptomycin. This study has provided a facile way to develop highly sensitive, effective and efficient aptamer-based electrochemical sensors for the detection of antibiotics at very low concentration.

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF‐κB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF‐κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF‐κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1‐ and K63‐linked ubiquitin chains are generated. NF‐κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria‐nucleus contact sites in a HOIP‐dependent manner. Notably, TNF‐induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1‐ubiquitin‐specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF‐mediated NF‐κB activation, both serving as a signaling platform, as well as a transport mode for activated NF‐κB to the nuclear.