微量蛋白的快速檢測在食品安全、臨床生物標記監測、量化細胞內蛋白質表現和檢測微量細菌毒素的相關領域中至關重要。當前臨床應用上，對快速、靈敏的生物標記物的檢測需求不斷增加，特別是對於早期疾病診斷、治療反應的即時監測和個人化醫療領域，更是備受關注，而在細菌毒素相關領域中，肉毒桿菌是引起神經麻痺疾病的最致命的細菌毒素之一，其非常微量就能致命，由上述內容可知，當前對有毒蛋白質和其他臨床相關生物標記物的檢測工具的迫切需求。為了滿足這些需求，我們提出了一種基於珠子的奈米免疫感測器，此感測器是基於擴散技術進行檢測，可快速、直接且精確地檢測生物毒素、臨床生物標記和其他感興趣的蛋白質。
我們設計的奈米免疫感測器是透過在螢光探針顆粒上連接包含金奈米顆粒 (AuNP)、目標蛋白和抗體的夾層免疫複合物。核心原理是，隨著更多的目標蛋白與其結合，整體免疫複合物顆粒的擴散率會隨之降低，這個研究在許多複雜基質（例如牛奶和牛血清）中建立了純化肉毒桿菌毒素（0.01–500 ng/mL）的校準曲線，表明此奈米免疫感測器的測量可有效的避免背景雜訊干擾。
除了檢測完整的毒素外，奈米免疫感測器還可以透過用突觸體相關蛋白 25 (SNAP-25) 等不同蛋白包被探針顆粒來評估毒素活性。當活性肉毒桿菌毒素剪切 SNAP-25 後，AuNP-抗體綴合物與其結合，導致擴散度降低，由此可應用於檢測毒素活性。值得注意的是，這種基於擴散的方法在 2 分鐘時間內實現了低至10 pg/mL的檢測極限。
此奈米免疫感測器具有快速、靈敏和不依賴基質的特性，可以方便地進行現場篩檢，以保護食品供應、保持與驗證醫療器械和化妝品的安全性以及檢測其他重要的臨床生物標記。這種創新的奈米免疫感測平台在快速識別複雜環境和臨床樣本中微量蛋白的檢測需求方面；具有廣闊的應用前景。

Rapidly detecting trace amounts of proteins is essential for ensuring food safety, monitoring clinical biomarkers, quantifying intracellular protein expression, and detecting bacterial toxins. In clinical settings, the demand for rapid and sensitive biomarker detection is ever-increasing, particularly for early disease diagnosis, treatment monitoring, and personalized medicine. Clostridium botulinum, one of the deadliest bacterial toxins causing neuroparalytic disease, further highlights the urgent need for appropriate detection tools for toxic proteins and other clinically relevant biomolecules. In response to these demands, we present a bead-based nano-immunosensor that operates based on the diffusometric technique for the quick, straightforward, and precise detection of biological toxins, clinical biomarkers, and other proteins of interest.
This nano-immunosensor was developed by creating sandwiched immunocomplexes involving gold nanoparticles (AuNPs), target proteins, and antibodies on fluorescent probe particles. The core principle is that the diffusivity of particles decreases as more target proteins bind to them. Calibration curves were successfully generated for purified botulinum toxins (0.01–500 ng/mL) in complex matrices such as whole milk and bovine serum, indicating that the measurement is not affected by background interference.
In addition to detecting intact toxins, the nano-immunosensors can also assess toxin activity by coating the probe particles with a substrate protein like synaptosomal-associated protein 25 (SNAP-25). When active botulinum toxin cleaves SNAP-25, AuNP-antibody conjugates bind to it, causing a slight reduction in diffusivity to indicate toxicity. Notably, this diffusion-based method achieves detection limits as low as 10 pg/mL within a 2 min timeframe.
The rapid, sensitive, and matrix-independent characteristics of these nano-immunosensors allow for convenient onsite screening to protect food supplies, maintain hygiene in medical instruments, verify the safety of cosmetic products, and detect other crucial biomarkers. This innovative biosensing platform holds great promise in meeting the increasing societal demand for rapid identification of trace proteins in complex environmental and clinical samples.

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