Plasmonic metal nanoparticles as efficient mass tags for ion signal amplification and ultrasensitive detection of protein markers

Highlights

• Efficient utilization of LSPR property of noble metal nanoparticle for generating abundant mass reporter ions.

• Plasmonic metal nanoparticle as ion reservoir for MS signal amplification.

• Specific and sensitive detection of high-mass protein in complex biological sample.

Abstract

The development of sensitive and specific analytical methods is critical for the discovery of molecular biomarkers, which assists disease diagnosis and understanding biological processes. Herein, a highly sensitive method is developed using antibody-conjugated plasmonic metal nanoparticles for the detection of targeted biomarkers down to low attomole level via coupling of immunoassay techniques with laser ionization mass spectrometry (LI-MS). The conjugated antibodies target specific antigens, while the metal nanoparticles act as mass tags and ion reservoirs for the signal amplification. With the characteristic localized surface plasmon resonance (LSPR) properties, gold (AuNPs) and silver nanoparticles (AgNPs) undergo explosive ionization upon laser irradiation to generate abundant characteristic mass reporter ions for strong MS signal amplification. With the antibody-conjugated NPs, detection of trace proteins in various biological samples with complex matrix environment, including urine, cell lysates, and animal tissues was demonstrated.

Introduction

Proteomics is of prime importance in comprehensive understanding of biological systems and plays a key role in disease diagnosis, drug development, and biomarker discovery [[1], [2], [3]]. Its success is highly dependent on the development of sensitive and specific methods for analyzing trace proteins with wide mass range in complex biological environment [4,5]. Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) is an essential analysis method for proteomics, especially for high-throughput analysis and intact biological tissue analysis [6,7]. Despite significant advancements in MALDI-MS technology in the past decades, high-mass protein analysis remains challenging [8,9]. In practice, proteins with molecular mass greater than 50 kDa, are relatively less sensitive to be detected even at microgram level [10], attributed to decreased ionization and detection efficiencies. In addition, with more than 45% of human proteins with mass larger than 50 kDa [11], the constraint of MALDI-MS limits the variety of proteins that could be analyzed. Thiery and co-workers developed an organic photo-cleavable mass tags linked with antibodies for targeting specific proteins [12,13]. The mass tag ions can be liberated by laser irradiation and are subsequently detected by the mass spectrometer, which indicates the presence of target proteins. Caprioli and co-workers further developed a PAMAM dendrimer as a mass tag container, which holds 900 mass tags for enhancing the sensitivity of the mass tag approach [14]. Apart from organic mass tags, heavy metal ions were also implemented as mass reporters in mass cytometry [15] and secondary ion mass spectrometry (SIMS) [16] in which multiple antigens were tagged and reported by individual metal ions. Nonetheless, the relatively low sensitivity of the methods is a major drawback [17], probably limited by the number of metal ions chelated onto the antibodies.

In our previous studies on the development of noble metal nanoparticles (NPs) as substrates for Surface-assisted Laser Desorption/Ionization Mass Spectrometry (SALDI-MS) [18,19], we consistently observed that under mild laser irradiation, abundant metal ions, such as Au⁺/Au₂⁺ and Ag⁺/Ag₂⁺ could be generated from AuNPs and AgNPs respectively [20,21]. This phenomenon could be due to the distinctive localized surface plasmon resonance (LSPR) properties of noble metal NPs [22], which enabled strong absorption of laser photo energy for efficient generation of electron-hole pairs on the surface [23], followed by the subsequent Columbic explosion to generate abundant metal ions [21]. Moreover, we particularly noted that being different from the organic mass tags, a AuNP can act as an ion reservoir for reporter ion generation. For instance, a single AuNP of 20 nm already contains approximately 2.5 × 10⁵ Au atoms. This reporter ion amplification factor is unlikely achievable by organic mass tags. As a result, we decided to conjugate AuNPs or AgNPs with antibodies for targeting high-mass proteins, and utilize the efficient laser-induced ion generation and high atom density properties of AuNPs/AgNPs for the protein detection and signal amplification.

While Zhang and co-workers previously adopted antibody-conjugated AuNPs as mass tag for the analysis of protein standards on microarray [24], their work using laser ablation inductively coupled plasma (LA-ICP) MS method employed a highly intense laser fluence (∼2 × 10⁴ J/cm²) to trigger energetic laser ablation processes, for non-selective breakdown and vaporization of sample materials that were subsequently transferred by a helium carrier gas to ICP for ionization. In contrast, our current approach takes the advantage of the LSPR property of plasmonic metal NPs which enables the adoption of a much milder laser fluence (at ∼ 2 × 10⁻¹ J/cm²) for selectively and directly generating Au reporter ions from the NPs. The mild laser fluence allows repetitive laser excitation and layer-by-layer ionization of the NPs for more efficient utilization of the NPs in ion signal amplification. Moreover, the mild laser fluence can also suppress the formation of background interfering ions to achieve higher signal-to-noise ratio, thus enabling specific and ultrasensitive detection of target molecules in biological fluids and tissues. The working principle of immunocapture laser ionization mass spectrometry (LI-MS), which makes use of the dual-function properties of antibody-conjugated plasmonic metal NPs, is shown schematically in Fig. 1a.